Vincent Wv Jaddoe
email: v.jaddoe@erasmusmc.nl

Cock M Van Duijn

Albert J Van Der Heijden

Johan P Mackenbach

Henriëtte A Moll

Eric Ap Steegers

Henning Tiemeier

Andre G Uitterlinden

Frank C Verhulst

Albert Hofman

THE GENERATION R STUDY: DESIGN AND COHORT UPDATE 2010

The Generation R Study is a population-based prospective cohort study from fetal life until young adulthood. The study is designed to identify early environmental and genetic causes of normal and abnormal growth, development and health during fetal life, childhood and adulthood. The study focuses on four primary areas of research:

(1) growth and physical development; (2) behavioural and cognitive development;

(3) diseases in childhood; and (4) health and healthcare for pregnant women and children. In total, 9,778 mothers with a delivery date from April 2002 until January 2006 were enrolled in the study. General follow-up rates until the age of 4 years exceed 75%. Data collection in mothers, fathers and preschool children included questionnaires, detailed physical and ultrasound examinations, behavioural observations, and biological samples. A genome wide association screen is available in the participating children. Regular detailed hands on assessment are performed from the age of 5 years onwards. Eventually, results forthcoming from the Generation R Study have to contribute to the development of strategies for optimizing health and healthcare for pregnant women and children.

INTRODUCTION

The Generation R Study is a population-based prospective cohort study from fetal life until young adulthood. The study is designed to identify early environmental and genetic causes of normal and abnormal growth, development and health during fetal life, childhood and adulthood. The study focuses on four primary areas of research:

(1) growth and physical development; [START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF] behavioural and cognitive development; [START_REF] Jaddoe | Fetal nutritional origins of adult diseases: challenges for epidemiological research[END_REF] diseases in childhood; and (4) health and healthcare for pregnant women and children. The background and specific lines of investigation have been described in detail previously [START_REF] Hofman | Growth, development and health from early fetal life until young adulthood: the Generation R Study[END_REF][START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF][START_REF] Jaddoe | Fetal nutritional origins of adult diseases: challenges for epidemiological research[END_REF]. The main outcomes and determinants are presented in Tables 1 and2. The general aims of the study are: [START_REF] Hofman | Growth, development and health from early fetal life until young adulthood: the Generation R Study[END_REF] To describe normal and abnormal growth, development and health from fetal life until young adulthood; [START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF] To identify biological, environmental and social determinants of normal and abnormal growth, development and health from fetal life until young adulthood; [START_REF] Jaddoe | Fetal nutritional origins of adult diseases: challenges for epidemiological research[END_REF] To develop and evaluate strategies for prevention and early identification of groups at risk. Special interest is in exploring the role of early environmental exposures and genetic variants in pathways leading to common diseases during fetal life, childhood and adulthood, of which various examples have been published in this journal [START_REF] Lawlor | Association of childhood intelligence with risk of coronary heart disease and stroke: findings from the Aberdeen Children of the 1950s cohort study[END_REF][START_REF] Lawlor | Associations of birth size and duration of breast feeding with cardiorespiratory fitness in childhood: findings from the Avon Longitudinal Study of Parents and Children (ALSPAC)[END_REF][START_REF] Nguyen | Influence of childhood parental history of type 2 diabetes on the pre-diabetic and diabetic status in adulthood: the Bogalusa Heart Study[END_REF][START_REF] Osler | The influence of birth weight and body mass in early adulthood on early coronary heart disease risk among Danish men born in 1953[END_REF][START_REF] Pearce | Sex differences in the association between infant feeding and blood cholesterol in later life: the Newcastle thousand families cohort study at age 49-51 years[END_REF][START_REF] Rask | Functional somatic symptoms and associated impairment in 5-7-year-old children: the Copenhagen Child Cohort 2000[END_REF][START_REF] Rzehak | Period-specific growth, overweight and modification by breastfeeding in the GINI and LISA birth cohorts up to age 6 years[END_REF][START_REF] Alatupa | School performance as a predictor of adulthood obesity: a 21-year follow-up study[END_REF][START_REF] Fraga | Children at danger: injury fatalities among children in San Diego County[END_REF][START_REF] Ghasemi | Nitric oxide and clustering of metabolic syndrome components in pediatrics[END_REF][START_REF] Heys | Is childhood meat eating associated with better later adulthood cognition in a developing population?[END_REF][START_REF] Huisman | Cognitive ability, parental socioeconomic position and internalising and externalising problems in adolescence: Findings from two European cohort studies[END_REF].

Main outcomes include risk factors for cardiovascular disease, type 2 diabetes, obesity, asthma, psychopathology and riks factors such as impaired physical activity . Results forthcoming from the Generation R Study have to contribute to the development of strategies for optimizing health and healthcare for pregnant women and children.

STUDY AREA

The Generation R Study is conducted in Rotterdam, the second largest city in the Netherlands. Rotterdam is situated in the Western part of the Netherlands on almost 80 km south from Amsterdam, the capital of the Netherlands. The total population consists of about 600,000 inhabitants of almost 150 different ethnicities. The study area is well defined by postal codes and covers more than half of the cities inhabitants (almost 350,000 inhabitants) [START_REF] Wiesbauer | Elevated risk of myocardial infarction in very young immigrants from former Yugoslavia[END_REF]. The largest ethnic groups in this population are the Dutch (56%), Surinamese (9%), Turkish (7%), Moroccan (6%), Dutch Antillean (3%) and Cape Verdian (3%) groups [START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF]. The percentages of the non-Dutch groups are higher in younger age groups [START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF]. The number of children born in this study area is about 4,300 per year. Measurements in the prenatal phase of the study were conducted in two well-equipped research centers in the study area, with a close collaboration with midwives and hospitals. In the preschool period, detailed measurements were conducted in a dedicated research center in the Erasmus Medical Center -Sophia Children's Hospital and routine care data were collected in five hospitals and sixteen child health centers located in this area.

STUDY DESIGN

Overview

The Generation R Study is a population-based prospective cohort study from fetal life until young adulthood. Mothers with a delivery date between April 2002 and January 2006 were eligible. Extensive assessments have been carried out in mothers and fathers and are currently performed in their children (Table 3 andTable 4).

Assessments in pregnancy were planned in early pregnancy (gestational age <18 weeks), mid-pregnancy (gestational age 18-25 weeks) and late pregnancy (gestational age >25 weeks). These measurements are considered as first, second and third trimester measurements. The fathers were assessed once in pregnancy.

The children form a prenatally recruited birth-cohort that will be followed until young adulthood. In the preschool period, which in the Netherlands refers to the period from birth to the age of four years, data collection was performed by a home-visit at the age of 3 months, questionnaires at the ages of 2, 6, 12, 18, 24, 30, 36 and 48 months and visits to the routine child health centres at the ages [START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF][START_REF] Jaddoe | Fetal nutritional origins of adult diseases: challenges for epidemiological research[END_REF][START_REF] Lawlor | Association of childhood intelligence with risk of coronary heart disease and stroke: findings from the Aberdeen Children of the 1950s cohort study[END_REF][START_REF] Nguyen | Influence of childhood parental history of type 2 diabetes on the pre-diabetic and diabetic status in adulthood: the Bogalusa Heart Study[END_REF][START_REF] Alatupa | School performance as a predictor of adulthood obesity: a 21-year follow-up study[END_REF][START_REF] Heys | Is childhood meat eating associated with better later adulthood cognition in a developing population?[END_REF][START_REF] Turin | Incidence, admission and case-fatality of acute myocardial infarction: weekend versus weekday in a Japanese population: 16-year results from Takashima AMI Registry (1988-2003)[END_REF][START_REF] Niemiec | The M235T polymorphism of the AGT gene modifies the risk of coronary artery disease associated with the presence of hypercholesterolemia[END_REF][START_REF] Greiser | Cardiovascular diseases, risk factors and short-term heart rate variability in an elderly general population: the CARLA study 2002-2006[END_REF]36 and 45 months (Table 4). Additional, more detailed assessments of fetal and postnatal growth and development have been conducted in a randomly selected subgroup of Dutch children and their parents at a gestational age of 32 weeks and postnatally at the ages of 1.5, 6, 14, 24, 36 and 48 months (Table 3 andTable 4).

Dutch is defined as two parents and four grandparents born in the Netherlands.

From the age of 5 years onwards, regular detailed hands on assessment are performed in all children and their parents in a well-equipped, dedicated research center.

Eligibility and enrolment

Eligible mothers were those who were resident in the study area at their delivery date and had a delivery date from April 2002 until January 2006. We aimed to enroll mothers in early pregnancy (gestational age <18 weeks) but enrolment was allowed until birth of their child. Midwives and obstetricians informed eligible mothers about the study at their first prenatal visit in routine care, handed out the information package and asked these mothers to make an appointment for the first ultrasound examination. The study staff contacted these mothers by phone for additional information about the study and in person at the ultrasound examination to obtain informed consent. Mothers who were not approached in pregnancy, were approached and enrolled in the first months after birth of their child when newborns visit the routine child health centers [START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF]. The fathers were not approached directly by the study staff but the mothers were informed about the importance of involvement of the fathers in the study.

STUDY COHORT

Parents

In total, 9,778 mothers were enrolled in the study (Figure 1). Of these mothers, 91% (n = 8,880) was enrolled in pregnancy. Only partners from mothers enrolled in pregnancy were invited to participate. In total, 71% (n = 6,347) of all fathers was enrolled. The general characteristics of the mothers and fathers are presented in Table 5. Of all participating mothers, enrolment was in early pregnancy in 69% (n = 6,691), in mid-pregnancy in 19% (n = 1,918), in late pregnancy in 3% (n = 271) and at birth of their child in 9% (n = 898). Of all pregnant women enrolled, 94% (n = 8,356), 6% (n = 516) and 0.1% (n = 8) were first, second and third pregnancies in the study, respectively. A total of 1,232 pregnant women and their children were enrolled in the subgroup for additional detailed studies. Ethnicity of participating mothers and partners was defined according the classification of Statistics Netherlands [71,[START_REF]Allochtonen in Nederland 2004[END_REF].

The largest ethnic groups were the Dutch, Surinamese, Turkish and Moroccan groups. The ethnic distribution differed only moderately from that of the population in the study area [71]. Mean household income in Rotterdam is about €1600 per month and the percentage subjects with a secondary or higher education level in Rotterdam is 56% [71]. The educational level of participating mothers and their partners was classified in groups according to the classification of Statistics Netherlands [START_REF] Troe | Explaining differences in birthweight between ethnic populations. The Generation R Study[END_REF].

Ethnic background, educational level and occupational status are of major interest and are studied as determinants of health and behavioural outcomes [74][START_REF] Troe | Smoking during pregnancy in ethnic populations: the Generation R study[END_REF][START_REF] Chote | Explaining Ethnic Differences in Late Antenatal Care Entry by Predisposing, Enabling and Need Factors in the Netherlands. The Generation R Study[END_REF][START_REF] Chote | Ethnic differences in antenatal care use in a large multi-ethnic urban population in the Netherlands[END_REF][START_REF] El Marroun | Demographic, emotional and social determinants of cannabis use in early pregnancy: the Generation R study[END_REF][START_REF] Timmermans | Determinants of folic acid use in early pregnancy in a multi-ethnic urban population in The Netherlands: the Generation R study[END_REF][START_REF] Van Rossem | Breastfeeding patterns among ethic minorities: The generation R study[END_REF][START_REF] Silva | No midpregnancy fall in diastolic blood pressure in women with a low educational level: the Generation R Study[END_REF][START_REF] Silva | Low socioeconomic status is a risk factor for preeclampsia: the Generation R Study[END_REF][START_REF] Silva | Maternal educational level and risk of gestational hypertension: the Generation R Study[END_REF][START_REF] Silva | Mother's educational level and fetal growth: the genesis of health inequalities[END_REF][START_REF] Jansen | Explaining educational inequalities in preterm birth: the generation r study[END_REF][START_REF] Jansen | Explaining educational inequalities in birthweight: the Generation R Study[END_REF][START_REF] Jansen | Employment status and the risk of pregnancy complications: the Generation R Study[END_REF][START_REF] Jansen | Socioeconomic inequalities in infant temperament: the generation R study[END_REF][START_REF] Jansen | National Origin and Behavioural Problems of Toddlers: The Role of Family Risk Factors and Maternal Immigration Characteristics[END_REF][START_REF] Van Rossem | Are starting and continuing breastfeeding related to educational background? The generation R study[END_REF][START_REF] Van Rossem | Socioeconomic Status is not Inversely Associated with Overweight in Preschool Children[END_REF]. Both household income and highest followed educational level in mothers and fathers in the study cohort suggest a selection towards a higher socioeconomic status than in the whole study. This pattern is similar as in other large scale cohort studies [START_REF] Jacobsen | Selection by socioeconomic factors into the Danish National Birth Cohort[END_REF]. However, differences between the population and cohort characteristics may also be due to selective missing values of ethnicity and socio-economic status in the questionnaires. Socio-economic status is related to various perinatal and postnatal health outcomes and of major interest in the study [START_REF] Rodrigues | Maternal unemployment: an indicator of spontaneous preterm delivery risk[END_REF][START_REF] Quansah | Work as a physician and adverse pregnancy outcomes: a Finnish nationwide population-based registry study[END_REF][START_REF] Mcfadden | Occupational social class, risk factors and cardiovascular disease incidence in men and women: a prospective study in the European Prospective Investigation of Cancer and Nutrition in Norfolk (EPIC-Norfolk) cohort[END_REF][START_REF] Mcfadden | Different measures of social class in women and mortality[END_REF][START_REF] Mcfadden | How far can we explain the social class differential in respiratory function? A cross-sectional population study of 21,991 men and women from EPIC-Norfolk[END_REF][START_REF] Mcfadden | Occupational social class, educational level, smoking and body mass index, and cause-specific mortality in men and women: a prospective study in the European Prospective Investigation of Cancer and Nutrition in Norfolk (EPIC-Norfolk) cohort[END_REF][START_REF] Abatih | Variance estimation methods for health expectancy by relative socio-economic status[END_REF][100][101][102][103][104][105][106][107][108][109].

Children at birth and overall response

Among the live births, 51% were male and 49% female. These percentages are similar to the population figures in the Netherlands and in Rotterdam [71]. The percentages of children born preterm or with low birth weight are smaller than expected on the basis of the population figures. This seems to reflect a selection toward a relative more healthy study population. Estimation of the precise number of eligible pregnant women in the study area is difficult since there is no satisfactory registry of pregnancies. Therefore, it was not attempted to identify overall response rates of pregnant women. Since the children form a prenatally recruited birth-cohort, the overall response of the study has been established at birth and is 61%.

Participation in postnatal follow up studies

As described above, 9,778 mothers were enrolled in the study and gave birth to 9,745 known live born children. The logistics of the postnatal follow up studies were embedded in the municipal routine child care system and restricted to the study area.

In total 1,163 children lived outside this definite study area at birth and were therefore not approached for the postnatal follow up studies during the preschool period. Of the remaining 8,582 children, 689 (8%) mothers did not give consent for the postnatal phase of the study leaving 7,893 children for the postnatal follow-up studies.

Reasons for non-participation in the postnatal follow up studies were primarily time restrictions or plans for moving from the study area. Of the total of 7,893 mothers, 598 (7.5%) gave a restricted consent and did not want to participate in the questionnaire studies. In total 7588 (78%) children or their parents participated in at least one measurement during the preschool period, of whom 6845 (90%) completed at least one questionnaire.

DATA COLLECTION IN THE PRENATAL PHASE

Physical examinations

Physical examinations were planned at each visit in early pregnancy, mid-pregnancy and late pregnancy and included height, weight and blood pressure measurements of both parents. Overall response rates for these specific measurements in mothers and fathers are similar as the visit percentages presented in Figure 1. Since there was a wide range of gestational age at each visit, these measurements are used in the analyses as gestational age adjusted measurements [110].

Questionnaires

Mothers received four postal questionnaires and father received one postal questionnaire in the prenatal phase (Table 3). All questionnaires are available in three languages (Dutch, English and Turkish). If needed, further support for verbal translation of questionnaires is available in Arabic, Portuguese and French. Each questionnaire comprises about 25 pages and takes about 30 to 45 minutes to be completed [START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF]. Topics in these questionnaires were:

-Mother 1: medical and family history, previous pregnancies, quality of life, life style habits, housing conditions, ethnicity, educational level; -Mother 2: diet, including macronutrients and micronutrients; -Mother 3: current pregnancy, quality of life, life style habits, psychopathology; -Mother 4: current pregnancy, quality of life, life style habits, working conditions, household income, self-esteem; -Father: medical history, family history, life style habits, educational level, psychopathology.

Overall response rates for these questionnaires varied from 77 to 91% (Figure 1).

However, the response rates of specific questions may be lower due to missing values within questionnaires.

Fetal ultrasound examinations

Fetal ultrasound examinations were performed at each prenatal visit. Overall response rates for these ultrasound examinations were in general similar to the visit percentages given in Figure 1. These ultrasound examinations were used for both establishing gestational age and assessing fetal growth patterns. These methods have previously been described in detail [111,112]. Establishing gestational age by using the first day of the last menstrual period is not reliable for a variety of reasons including the large number of women who do not know their exact date, have irregular menstrual cycles or amenorrhea, use oral contraceptive pills or bleed in early pregnancy [113]. Using fetal ultrasound data such as crown-rump length or biparietal diameter for pregnancy dating seems to overcome these problems but does not allow growth studies of these measurements since no growth variability between subjects is assumed. Pregnancy dating-curves have been derived in a subsample of the cohort including subjects with complete data on both the first day of the last menstrual period and crown-rump length or biparietal diameter and used to date the gestational age [111]. Subsequently, longitudinal curves of all fetal growth measurements (head circumference, biparietal diameter, abdominal circumference and femur length) were created resulting in standard deviation scores for all of these specific growth measurements. Various sociodemographic and lifestyle related determinant seems to affect these fetal growth and birth outcomes [114][115][116][117][118][119]. Also, specific fetal growth patterns seem to be associated with outcomes in childhood

Pregnancy complications and outcomes

The obstetric records of mothers have been looked up in the hospitals and mid-wife practices. Specialists in the relevant field code items in these records, and used for validation studies for maternal reported data [130,131]. The major pregnancy outcomes, including live births, induced abortion fetal or perinatal loss, pregnancy induced hypertension, preeclampsia, and gestational diabetes are known in 99% of all enrolled mothers. These outcomes are related to various exposures of interest [132][133][134][135][136][137][138][139][140][141][142][143][144][145][146][147][148]. In all children known to be born alive, information about sex, birth weight and gestational age is available. Currently, efforts are made to link the Generation R dataset to existing national databases for pregnancy and the neonatal period [149,150].

Biological samples

Blood samples were collected in early and mid-pregnancy and at birth. Procedures for collection, processing and storage of biological samples have been described previously in detail [151]. The planned amounts of venous blood taken were 35 and 20 ml in early pregnancy and mid-pregnancy, respectively, from the mother and 10 ml from father. At delivery, 30 ml cord blood was collected. Blood samples are currently available for 97%, 83% and 67% of the participating mothers, partners and children, respectively. Due to several clinical complications in mother and child, it was not always possible to collect cord blood. Subsequently, availability rates of cord blood samples in children born preterm or with low birth weight are lower than expected. Plasma and serum samples has been distributed in small aliquots (each 250 μl) and stored at -80 o C. Maternal and cord blood samples have been used for measuring dietary biomarkers (folate, homocystein, total vitamin B12, free vitamin B12) levels; angiogenesis biomarkers (soluble fms-like tyrosine kinase-1 (sFlft-1), Placental growth factor (PlGF)); thyroid hormone levels (thyroid-stimulating hormone (TSH), free thyroxine (FT4)) and thyroid antibody levels, inflammation markers (highsensitivity C-reactive protein (hs CRP); and celiac disease and Helicobacter Pylori antibodies [152][153][154]. Urine samples of mothers have been collected from February 2004 until November 2005 and are stored for future measurements. Response rates for these urine samples are 85%, 97% and 96% in early, mid-and late pregnancy respectively. These urine samples are used for measurement of several environmental exposures, and metabolites. Urine samples have been used for measurement of Chlamydia, and pesticides, bisphenol A, and phthalates levels [155][156].

DNA and genome wide association studies

DNA from both parents (whole blood) and children (cord blood) has been extracted, normalised and plated and is currently used for several genotype studies. A genome wide association scan (GWAS) using the Illumina 610 Quad platform is available for the participating children. For genotyping, we used the infrastructure of the Genetic Laboratory of the Department of Internal Medicine (www.glimdna.org) that was also used for creation of the GWAS datasets of the Rotterdam Study, a prospective cohort study among more than 10,000 adults [157,158]. The GWAS dataset underwent a stringent QC process whereby Individuals with low call rates or sex mismatches were excluded, and SNPs with low call rates were excluded. In addition, ethnic composition of the sample was estimated by Identity-By-State Analysis, using Principal Component Analysis, and cryptic familial relationships were identified through Identity-By-Descent analysis]. To maximize genome coverage and allow inter-study comparisons, we used MACH (version 1.0.15) software to impute genotypes at each of the 2.5 million autosomal CEPH HapMap II (release 22) SNPs for Europeans and European-Americans [159]. Criteria such as high levels of missing data (SNP call rate <98%), highly significant departures from Hardy-Weinberg equilibrium (p<1. , or low Minor Allele Frequencies (MAF<1%) were used to determine which SNPs to include in the imputation step. Most GWAS analyses are strongly embedded in the recently started Early Growth Genetics (EGG)Consortium and Early Genetics and Longitudinal Epidemiology (EAGLE) Consortium, in which several birth cohort studies combine their GWAS efforts focused on multiple outcomes in fetal life, childhood and adolescence [160,161]. The power of large scale consortia has been shown previously [162][163][164][165][166][167][168][169][170]. Also, strategies for genetic scoring analyses are being developed to estimate predictive values of identified genetic variants [171,172]. Although a GWAS is available in children, we use DNA from both children and parents for genotyping for candidate gene or replication studies [173][174][175][176][177][178][179][180][181][182]. Missing data in DNA samples in children will be collected by blood sampling or other methods at follow-up measurements.

DATA COLLECTION IN THE FIRST 4 POSTNATAL YEARS

Physical examinations

At the age of 3 months, home visits were performed to assess neuromotor development using an adapted version of Touwen's Neurodevelopmental examination [183][184][185]. Information about growth (length (height), weight, head circumference) is collected at each visit to the routine child health centres in the study area using standardized procedures. These visits are planned at the ages 2, 3, 4, 6, 11, 14, 18, 24, 30, 36 and 48 months. Response rates for these visits vary between 70 and 97% [186].

Questionnaires

During the preschool period, mothers receive 8 questionnaires and fathers receive 1 questionnaire. Each questionnaire comprises about 25 pages. Items included in these questionnaires and their references are demonstrated in table 5 [187-223].

Response rates based on the number of send questionnaires are showed in figure 1.

Due to logistical constraints and implementation of questionnaires after the first group of children reached a certain age, not all children received each questionnaire. Thus, although response rates may be similar, the absolute number of completed questionnaires could differ between different ages. Response rates presented in figure 1 are based on the number of send questionnaires.

Hands on assessments and observations in subgroup

During the preschool period, children participating in the subgroup studies have been invited six times to a dedicated research center. Measurements at these visits included physical examinations (height, weight, head circumference, skinfold thickness and waist-hip ratio, Touwen's Neurodevelopmental Examination at the ages of 1.5, 6, 14, and 24 months) and ultrasound examinations (brain structures at months, cortisol day rhythm measured by repeated salivary samples at the age of 14 months and, if parents give consent, blood samples at the ages of 6, 14 and 24 months [242-251]. Response rates for these blood samples are about 30%, mainly because of lack of parental consent.

Measurements from the age of 5 years

From the age of 5 years onwards, we plan to invite all participating children to a wellequipped and dedicated research center in the Erasmus Medical Center -Sophia Children's Hospital every three years. Currently, all children aged 5 to 6 years are invited to participate in hands on measurements, behavioural observations and biological sample collection. The total visit takes about 2.5 hours and all measurements are grouped in thematic 20 min blocks. These measurements are focused on several health outcomes including asthma, bacterial carriage, infectious diseases, behaviour and cognition, body composition, eye and tooth development, heart and vascular development, kidney growth and function, and obesity. These measurements will be used as both outcomes and determinants for health outcomes in later life. Cardiovascular, metabolic and bone measurements are also conducted in mothersClinically relevant results are discussed with the parents and, if needed, children or mothers are referred to their general practitioner, paediatrician or other relevant health care provider. All children are expected to have visited our research center in summer 2011. Currently ideas for follow up studies including detailed imaging studies using Magnetic Resonance Imaging (MRI), are discussed and tested in subgroups.

ETHICAL ISSUES

The general design, all research aims and the specific measurements in the Generation R Study have been approved by the Medical Ethical Committee of the Erasmus Medical Center, Rotterdam. New measurements will only be embedded in the study after approval of the Medical Ethical Committee. Participants are asked for their written informed consent for the four consecutive phases of the study (prenatally, birth to 4 years, 4-16 years, and from 16 years onwards). At the start of each phase, mothers and their partners received written and oral information about the study. Even with consent of the parents, when the child is not willing to participate actively, no measurements are performed.

FOLLOW UP AND RETENTION STRATEGIES

Thus far, loss to follow up seems to be limited. Major efforts are made to keep the children and parents involved in the study and to minimize loss to follow up to prevent bias in future studies. Several strategies have been implemented and are currently part of the study design:

-Addresses: new addresses of participants, which are known by the municipal health service are forwarded to the study staff; -Newsletters: participants receive four newsletters per year, in which several results of the study are presented and explained, questions of participants are answered and new research initiatives are presented; -Presents and discounts: all children who visit our research center receive small presents. Also, discount offers are regularly part of the newsletter; -Transport costs: all costs for transport and parking related to visits to the research center are paid by Generation R; -Reminders for questionnaires: when the questionnaire has not been returned within three weeks, a kind reminder letter is send to the parents. After six weeks, when the questionnaire has still not been returned, the parents receive a phone call. If necessary, help for completing the questionnaire is offered and the importance of filling in the questionnaire is explained once more during this phone call; -Individual feedback: if clinically relevant, all results of hands-on measurements are discussed with the parents at the visit. If necessary, follow up appointments with the general practitioner or pediatrician are planned; -Support for ethnic minorities: all study materials such as questionnaires, newsletters, website, and information folders are available in three languages (Dutch, English, and Turkish). Furthermore, staff from different ethnic minorities is available and able to verbally translate these materials into Arabic, French and Portuguese. With this, the study staff is able to communicate to all participants.

-Care-cases: children and parents who showed low response rates for different measurements, showed difficulties in completing questionnaires or require additional explanation or support are considered as care-cases. Care -cases have a more individual based approach and are pro-actively contacted by one dedicated member of the study staff.

New methods for contacting participants, which have been used in other more recently started studies, including use of internet, sms and e-mail, are currently being explored [252][253][254][255][256][257][258][259][260][261][262][263].

DATA MANAGEMENT AND PRIVACY PROTECTION

Data collected by measurements in the research centers are directly entered onto written forms and into the electronic database. Data collected by questionnaires are scanned and manually entered into an electronic database by a commercial bureau.

Random samples of all questionnaires are double checked by study staff members to monitor the quality of this manual data entry process. The percentage of mistakes is kept as low as possible and does not exceed 3% per questionnaire. Open text fields are entered into the electronic database exactly as they are filled in on the questionnaires. In a secondary stage, these open text fields are cleaned and coded by a specialist in the relevant field. All measurements are centrally checked by examination of the data including their ranges, distributions, means, standard deviations, outliers and logical errors. Data outliers and missing values are checked on the original forms and might be imputed and subsequent appropriate statisticial techniques are used [264][265][266][267][268][269][270][271][272][273][274][275]. The data of one specific measurement are only distributed for analyses after data collection and preparation is completed for that measurement for the whole cohort. Datasets needed for answering specific research questions are centrally built from different databases. All information in these datasets that enables identification of a particular participant (including identification number used for the logistics of the study, names and dates) is excluded before distribution to the researchers. The datasets for researchers include subject unique identification numbers that enable feedback about one subject to the data manager but do not enable identification of that particular subject.

STATISTICAL POWER

Due to expected missing values and loss to follow-up, most analyses in the study are not based on data in all subjects. Therefore, power calculations demonstrated in Tables 7 and8 are based on 7,000 subjects in the whole cohort and 700 subjects in the focus cohort. Table 8 demonstrates that for a normally distributed continuous outcome it is possible to detect with a type I error of 5% and a type II error of 20% (power 80%) a difference of 0.11 SD in the whole cohort and of 0.35 SD in the focus cohort if 10% of all subjects has the relevant exposure. Table 8 presents for dichotomous outcomes that with the same type I and II errors, it is possible to detect a relative risk of 1.39 in the whole cohort and of 2.48 in the focus cohort if 10% of all subjects has the relevant exposure and the 1 year incidence of the outcome of interest is 10%. Rates of most dichotomous environmental and genetic exposures in the Generation R Study are expected to vary generally between 10 and 20%. The presented power calculations are rather conservative since most studies will assess the effects of continuously instead of dichotomous measured exposures and studies may be focused on outcomes collected in more than only one year. Furthermore, the Generation R Study has a large number of measurements repeated over time, which may increase the accuracy of measuring the true underlying value and may thereby increase the statistical power for these measurements. The presented effect sizes are detectable relative risks with a type I error of 5% and a type II error of 20% (power 80%).

COLLABORATION

  [120-123]. We have demonstrated in a subgroup study among mothers with a known and reliable last menstrual period, that various life style related factors affect first trimester growth [124]. Placental haemodynamics including resistance indices of the uterine and umbilical arteries have been assessed in second and third trimester [125]. Detailed measurements of fetal brain, cardiac and kidney development have been performed in the subcohort [126-129].

1. 5

 5 months and cardiac and kidney structures at the ages of 1.5, 6 and 24 months, abdominal fat at the age of 24 months) [224-237]. Dual X Energy Absorptiometry (DXA) scanning was performed in a subgroup of children at the age of 6 months [238]. Similarly Fractional Exhaled Nitric Oxide (FeNO) has been measured in only 50% of all children at the ages of 6 and 24 months. Blood pressure was measured at the age of 24 months [239]. Observations of parent-child interaction and behaviour, such as executive function, heart rate variability, infant-parent attachment, moral development, and compliance with mother and child have been performed at the ages of 14, 36 and 48 months and with father and child at the age of 48 months [240-241]. Biological materials have been collected, including bacterial colonization measured by nasal-and nasopharyngeal swabs at the ages of 1.5, 6, 14 and 24

  The Generation R Study is conducted by several research groups from the Erasmus Medical Center in close collaboration with the Erasmus University Rotterdam and the Municipal Health Service Rotterdam area. Since the data collection is still ongoing and growing, the number of collaborating research groups in and outside the Netherlands is expected to increase in the coming years. The study has an open policy in regard to collaboration with other research groups. Request for collaboration should primarily be pointed to Vincent Jaddoe (v.jaddoe@erasmusmc.nl). These requests are discussed in the Generation R Study Management Team regarding their study aims, overlap with ongoing studies, logistic consequences and financial contributions. After approval of the project by the Generation R Study Management Team and the Medical Ethical Committee of the Erasmus Medical Center, the collaborative research project is embedded in one of the four research areas supervised by the specific principal investigator.

Table 3 . Assessments in mothers, fathers and their children in the prenatal phase

 3

	+ = Assessment in whole cohort.
	S = assessment only in subgroup.
	Early pregnancy: gestational age <18 weeks; mid-pregnancy: gestational age 18-25 weeks; late
	pregnancy: gestational age >25 weeks.

Table 4 . Assessments in mothers, fathers and children during the preschool period

 4

								Age (months)
		2	3	4	6	11	12	14	18	24	30	36	45	48
	Child
	Questionnaire (parent)		+		+		+		+	+	+	+		+
	Physical examination	+	+	+	+	+		+		+		+	+
	Ultrasound	S			S					S
	Airway inflammation				S					S
	Behavioural observation							S				S		S
	Bacterial carriage	S			S					S		S
	Blood sample				S					S
	Mother specific
	Questionnaire		+		+							+		S
	Interaction with child							S				S
	Father specific
	Questionnaire											+
	Interaction with child													S
	+ = Assessment in whole cohort.
	S = assessment only in subgroup.

Table 8 . Relative risks that can minimally be detected according to the prevalence of the exposure Incidence (1 year) of outcome of interest

 8

		Whole cohort (n = 7,000)		Focus cohort (n = 700)
	Proportion exposed	10%	5%	1%	10%	5%	1%
	50%	1.23	1.33	1.83	1.83	2.28	4.94
	25%	1.26	1.38	1.94	1.96	2.46	5.41
	10%	1.39	1.56	2.42	2.48	3.26	7.92
	5%	1.55	1.80	3.09	3.20	4.39	11.74
	1%	2.36	3.04	6.83	7.75	11.61	37.55

ACKNOWLEDGEMENTS

The Generation R Study is conducted by the Erasmus Medical Center in close collaboration with the School of Law and Faculty of Social Sciences of the Erasmus University Rotterdam, the Municipal Health Service Rotterdam area, Rotterdam, the Rotterdam Homecare Foundation, Rotterdam and the Stichting Trombosedienst & Artsenlaboratorium Rijnmond (STAR-MDC), Rotterdam. We gratefully acknowledge the contribution of children and parents, general practitioners, hospitals, midwives and pharmacies in Rotterdam. We thank Rachel Bakker, Claudia Kruithof and Dr Fernando Rivadeneira for their study coordination, data management, and GWAS analyses. The general design of Generation R Study is made possible by financial support from the Erasmus Medical Center, Rotterdam, the Erasmus University Rotterdam, the Netherlands Organization for Health Research and Development (ZonMw), the Netherlands Organisation for Scientific Research (NWO), the Ministry of Health, Welfare and Sport and the Ministry of Youth and Families. Vincent Jaddoe received an additional grant from the Netherlands Organization for Health Research and Development (ZonMw 90700303, 916.10159).

Table 1.

Main outcomes per research area

Growth and physical development

Main determinants

Biological determinants

Parental anthropometrics and blood pressure


Timothy J Littlewood
email: tim.littlewood@chch.ox.ac.uk

Rachel Clarke

Kate Stretton

Janine White

Rachel ; Clarke

Rachel T Clarke

Janine Warnick

Improving the immediate management of neutropenic sepsis in the UK: lessons from a national audit

Keywords: NEUTROPENIA, CHEMOTHERAPY, CLINICAL HAEMATOLOGY, ANTIBIOTICS neutropenic sepsis, febrile neutropenia, audit, protocol, management Running Title; A UK audit of neutropenic sepsis management

Recent disquiet at inadequacies in the immediate management of neutropenic sepsis in the UK led to a new, gold standard "door-to-needle" time for the administration of intravenous antibiotics of one hour. The aim of this audit was to identify whether that target is being met nationally, the potential barriers to its achievement, and concrete recommendations for how to overcome these. We also sought to establish the degree of regional heterogeneity in current local management protocols. Questionnaires were sent to haematologists across the UK to determine their unit's immediate management of patients presenting from the community with possible neutropenic sepsis. Local protocols and audits were also requested. Data covering 95 different hospitals were received, covering a combined catchment area of nearly 30 million people. There were marked regional inconsistencies in the definition of "neutropenic sepsis" and almost every aspect of its immediate management. Only 26% of audited patients (n=627) received intravenous antibiotics within the target time of one hour. Median door-to-needle times ranged from 30 minutes to 3 hours 44 minutes. Long delays of over 5 hours were not uncommon.

Introduction

Neutropenic sepsis is a well-known and life-threatening complication of bone marrow dysfunction and cytotoxic chemotherapy. Associated mortality rates range from 2 to 21% [START_REF] Smith | Update of recommendations for the use of white blood cell growth factors: an evidence-based clinical practice guideline[END_REF][START_REF] Herbst | Prophylactic antibiotics and G-CSF for the prevention of infections and improvement of survival in cancer patients undergoing chemotherapy (Protocol)[END_REF]. Sepsis in general is a time-dependent medical emergency in which early, goal-directed resuscitation and the urgent administration of broad-spectrum antibiotics have proven benefits on outcome [START_REF] Dellinger | Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008[END_REF][START_REF] Gaieski | Impact of time to antibiotics on survival in patients with severe sepsis or septic shock in whom early goal-directed therapy was initiated in the emergency department[END_REF].

A recent national investigation into avoidable patient deaths following chemotherapy identified serious short-comings in the management of neutropenic sepsis in the UK, including delayed administration of intravenous antibiotics (NCEPOD, 2008). In 2 response, the UK's National Chemotherapy Advisory Group published guidelines for improving the management of neutropenic sepsis (NCAG, 2009). These recommended a new "door-to-needle" time for antibiotic administration of one hour maximum, mirroring the international Surviving Sepsis Campaign's gold standard [START_REF] Dellinger | Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008[END_REF]. The National Institute for Clinical Excellence has since been commissioned to draft a national guideline for the prevention and best management of neutropenic sepsis.

Yet to date, neither the degree of regional variation in existing hospital guidelines for managing neutropenic sepsis, nor the extent to which local centres successfully meet their own standards, have been investigated at a national level. There are no national data on whether the one-hour target for neutropenic sepsis is being met, whether door-to-needle times vary significantly between hospitals (and, if so, why), or even whether speed of antibiotic delivery has a proven benefit for clinical outcome.

These questions are pertinent to all health professionals involved in acute hospital care. Evidence shows that much of the immediate assessment and treatment of patients presenting with neutropenic sepsis is delivered not by haematologists or oncologists, but by frontline emergency department staff and junior doctors on acute medical rotas (NCAG, 2009). In other words, sub-optimal management of neutropenic sepsis is not a specialist concern so much as a practical imperative to be addressed by every hospital with acute facilities to which patients may present. This paper aims to augment the existing evidence base by conducting the UK's first national audit of the immediate management of neutropenic sepsis in haematology patients. It seeks to identify:

1) the degree of regional heterogeneity in existing local management protocols, 2) the extent to which the one-hour door-to-needle target is being met nationally, 3) the potential barriers preventing the one-hour target from being translated into achievable practice, and concrete recommendations to overcome these.

Key questions include whether best practice may be identified and transferred and whether, in so doing, patient outcomes are modifiable. The collation and dissemination of local protocol and audit findings may act as a practical resource for hospital trusts, informing future policy and potentially improving clinical outcome.

Methods

MEDLINE and EMBASE were searched for English language papers, without date restriction, using the terms "neutropenic sepsis", "febrile neutropenia", "management", "treatment", "audit", "protocol", "guideline", "gold standard", "best practice" and their synonyms. All potentially relevant abstracts were retrieved and read in full.

All British Society for Haematology members were emailed a questionnaire regarding their unit's immediate management of patients presenting from the community with possible neutropenic sepsis. In addition, all specific reasons for delayed door-to-needle time -and all recommendations for overcoming these -identified in audits, protocols and completed questionnaires were collated.

We were unable to calculate p-values for the combined audit data, since all but one of the individual audits did not provide them, and only a small number of audits supplied individual patient data.

Results

(Edit 6) The literature search yielded a total of 1975 abstracts. 98 papers were judged relevant and reviewed in full. Their findings informed the study design, drafting of the questionnaire, and data analysis. Responses from 71 haematology departments were received. 10 of these were excluded since their data covered paediatric or non-UK centres. (Edit 1) Although the remaining 61 departments represent a relatively small proportion (28%) of the total number of haematology departments in the UK (n=220), respondents frequently supplied protocols and/or audits which covered multiple hospitals within a trust or region. Consequently, the data provided covered adult patients in 95 different hospitals across the UK (Table I). Two thirds of these were district general hospitals; the rest tertiary referral centres. Their combined catchment area was just under 30 million people (over half the UK adult population).

100% of respondents stated that they had a written protocol governing their management of neutropenic sepsis, and 75% that they had audited their practice against these local standards. We received protocols from 90% of respondents and audits from 44% of them. The total number of patients audited was 627, with a median number of patients per audit of 22 (range 6-71). (Edit 2) It was not made clear whether the numbers of patients audited per centre was a reasonable representation of the incidence of neutropenic sepsis in each centre. The majority of the audits (89%) examined practice in 2008, 2009 or 2010. Of the remainder, the least up-to-date audit occurred in 2006.

Definitions of "febrile" and "neutropenia"

Definitions of "febrile" and "neutropenia" were extracted from each protocol provided (n=55). They demonstrated marked regional heterogeneity, with a total of ten different definitions recorded (Table II).

"Neutropenia" was defined more consistently, with 67% of protocols using an absolute neutrophil count of <1•0 x 10 9 /l, 25% using <0•5 x 10 9 /l or <1•0 x 10 9 /l and expected to fall to <0•5 x 10 9 /l, and 8% using other values. One protocol did not define neutropenia numerically, instead providing a list of the types of patients likely to be at risk due to immunocompromise.

(Edit 7) Multiple definitions of "febrile" and "neutropenia" were also noted in the literature review, suggesting an international lack of consensus on how best to define neutropenic sepsis. The clinical usefulness of even the original diagnostic criteria for sepsis, severe sepsis and septic shock, which use a temperature of >38°C or <36°C [START_REF] Bone | Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee[END_REF], has been increasingly questioned [START_REF] Cohen | How I manage haematology patients with septic shock[END_REF].

Risk stratification

15% of protocols (n=8) advocated classifying patients with potential neutropenic sepsis into low and high risk categories. Of these, four protocols recommended prescribing oral, as opposed to intravenous antibiotics to low-risk patients, as defined by the Multinational Association for Supportive Care in Cancer (MASCC) Risk Index [START_REF] Klaterskey | The Multinational Association for Supportive Care in Cancer risk index: a multinational scoring system for identifying low-risk febrile neutropenic cancer patients[END_REF].

First-line empirical antibiotics 87% of protocols (n=48) recommended intravenous Tazocin and gentamicin as their firstline empirical antibiotics for patients thought to be neutropenic and septic. The remainder Prophylactic antibiotics 48% of respondents (n=29) confirmed in questionnaires that they prescribed prophylactic antibiotics to patients at risk of neutropenia. Oral ciprofloxacin was first line in all but two of these units.

Specific instructions given in protocols

Protocols were screened for any specific instructions relating to the immediate assessment and management of patients which might influence outcome. Four such instructions were identified, and their prevalence noted (Table III).

In addition, respondents were asked in the questionnaire whether their unit recommended writing the first dose of antibiotics on the statim side of the drug chart, in order potentially to hasten their administration. 52% of units did so.

Time and location of initial presentation

Of the 27 audits received, 16 provided data on where patients initially presented (n=359). 59% of patients first presented to the emergency department (ED), to some form of acute medical assessment unit (MAU) or, rarely, to a high dependency unit (HDU). 41% presented directly to a specialist haematology or oncology ward.

Five audits (n=96) also documented the time of presentation. 50% of patients presented during core hours, defined as Monday -Friday, between 9am and 5pm. 50% presented out-of-hours, whether overnight or during weekends.

Symptoms on admission

Five audits noted patient signs and symptoms on presentation. Of these, three (n=82) documented patients' fever status. 70% of patients were febrile, 30% afebrile. One audit (n=71) noted that 15% of patients were asymptomatic on presentation, while 11% had a systolic blood pressure less than 90mm/Hg. In another small audit (n=6), 100% of patients had two or more signs of infection such as temperature, rigors or hypotension.

Time to first assessment 11 audits (n=137) described times to first nurse triage and/or to first doctor's assessment.

The median times to triage ranged from 10 min to 1 h. The longest single time to triage noted was 3 h 8 min. The median times to first doctor's assessment ranged from 1 h 40 min to 2 h. The longest period before a doctor's assessment was 12 h 38 min.

Effect of time and location of presentation on door-to-needle times

Six audits (n=104) examined whether where patients initially presented influenced speed of antibiotic delivery. In all six, the median door-to-needle time was longer when patients first presented to an ED/MAU rather than a Haematology/Oncology ward (Fig 2). In one study, the median door-to-needle time was eight times as long in the ED/MAU as it was on the day ward (4 h v 30 min).

Two audits (n=35) evaluated the impact of time of presentation on door-to-needle times.

In both, core-hours were defined as 9am-5pm, Monday to Friday; out-of-hours as weekends and weekday evenings/nights. The two median door-to-needle times for corehours presentations were 2 h 25 min and 1 h 30 min. For out-of-hours presentations, these increased to 2 h 54 min and 3 h min respectively.

(Edit 3) The small sample sizes precluded analysis of the extent to which time and location of initial presentation independently influence door-to-needle times, or are instead variables which confound each other.

Patient outcomes

Four audits (n=149) provided data on deaths of audited patients. The combined mean mortality rate was 9%.

Specific reasons identified for delayed door to needle time, and practical recommendations for overcoming these

Multiple different reasons for delayed door-to-needle times were identified in protocols, audits and completed questionnaires, such as antibiotics being prescribed in A&E but not given until transfer to a specialist ward, and doctors awaiting the full blood count before prescribing antibiotics. All such reasons are collated below (Table IV), organised into the following four categories:

• lack of staff • lack of understanding of neutropenic sepsis • practical problems • communication issues between different members of staff/teams (Edit 4) The five most commonly cited reasons for delay, in order of frequency, were:

1) Antibiotics being prescribed by doctor but their administration delayed by nurse, or until patient was transfered to specialist ward (20% of all reasons cited) 2) Prolonged time to assessment by junior doctor (13%) 3) Lack of awareness of natural evolution of course of neutropenic sepsis, particularly how quickly an apparently well patient can deterioriate (10%) 4) Emergency departments not stocking the appropriate intravenous antibiotics (7%) All practical recommendations for improving door-to-needle times described in protocols, audits and completed questionnaires are also listed (Table V). (Edit 5) 41% of audited departments (n=11) confirmed that the process of audit had led to improved doorto-needle times, citing the positive impact of specific innovations such as writing up antibiotics on the statim side of drug charts, ensuring emergency departments stocked appropriate antibiotics, and introducing patient-group directives for antibiotic prescription by nurses. The remaining audited departments made no mention of whether audit improved future performance.

Discussion

Although neutropenic sepsis is a common, costly and not infrequently fatal complication in haematology and oncology, it lacks clear, national, evidence-based guidelines informing urgency of antibiotic administration. In contrast, the immediate management of other lifethreatening, medical emergencies such as acute coronary syndrome and meningococcal sepsis tends to be deeply ingrained in everyday practice. For example, aspirin in the former and intramuscular antibiotics in the latter, have often been given before the patient even arrives in hospital.

One diagnostic challenge is that in immunosuppressed patients, the early presenting features of sepsis can be muted or even absent [START_REF] De Naurois | Management of febrile neutropenia: ESMO clinical practice guidelines[END_REF]). Yet for other acute illnesses whose initial presentation is highly non-specific, such as meningococcal sepsis in infants, a culture of erring on the side of caution and treating empirically exists precisely to avoid delayed diagnoses and their potentially catastrophic consequences [START_REF] Visintin | Management of bacterial meningitis and meningococcal septicaemia in children and young people: summary of NICE guidance[END_REF]. Our audit highlights the fact that in the UK neutropenic sepsis lacks even consistency of definition, let alone treatment, with both "neutropenia" and "febrile" subject to multiple interpretations across different trusts and regions. As for national consensusbased therapy, the limitations of the current evidence base offer scant support for robust guidelines. For although the benefits of a one-hour door-to-needle time in septic shock and severe sepsis are underpinned by research [START_REF] Dellinger | Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008[END_REF][START_REF] Gaieski | Impact of time to antibiotics on survival in patients with severe sepsis or septic shock in whom early goal-directed therapy was initiated in the emergency department[END_REF][START_REF] Kumar | Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock[END_REF], to the best of our knowledge no papers have examined its impact on neutropenic sepsis specifically, other than one small study of a two-hour target in intensive care unit patients, only half of whom were neutropenic [START_REF] Larche | Improved survival of critically ill cancer patients with septic shock[END_REF].

This study contributes to the evidence base by demonstrating that nationally, an aspirational door-to-needle time of one hour is far from being met in practice, with barely a quarter of audited patients receiving their antibiotics within this time. Our findings underscore the idea that late recognition of neutropenic sepsis might exacerbate delays, since in one local audit, nearly a third of patients were afebrile on presentation, while in another, 15% were asymptomatic. However, the fact that a small minority of hospitals (n=2) have managed to achieve the median target door-to-needle time suggests that such an aspiration is nonetheless achievable, and how these centres have achieved it demands further enquiry. Our audit data indicate that both the timing and the location of initial presentation may be relevant, since out-of-hours presentations and those to non-specialist admitting areas such as EDs or MAUs, show significantly increased door-to-needle times. Whether this relates primarily to greater staff-patient ratios, better understanding and awareness of neutropenic sepsis or increased familiarity with local protocol in specialist areas is not known. What is clear from the prolonged overall median times noted to first nurse triage (10 min -1 h) and to first doctor's assessment (1 h 40 min -2 h), is the current unfeasibility of improving compliance with the one-hour target.

In addition to staffing issues, local trusts have identified many other specific reasons for delay, with poor understanding of the natural history of neutropenic sepsis featuring prominently. This cannot be aided by local protocols described by respondents as inaccessible, hard to locate, or long and impenetrable to read. With this in mind, we screened all protocols received for clear, specific instructions which might promote shorter door-to-needle times, such as not waiting for a full blood count before giving antibiotics.

The prevalence of the four such instructions identified (Table 3) ranged from 71% to only 39%. Perhaps unsurprisingly then, junior doctors were cited by respondents as contributing to delays by waiting for blood counts, renal function, senior review and haematology opinion before prescribing antibiotics.

More encouragingly, many respondents identified the act of audit itself as a factor in improving their own standards, a phenomenon mirrored in the literature [START_REF] Amado | Effect of a quality improvement intervention to decrease delays in antibiotics delivery in pediatric febrile neutropenia: a pilot study[END_REF]. In addition to re-writing protocols and educating staff, hospitals have responded to identified delays by introducing innovations as diverse as patient-held alert cards with a pre-signed first dose of antibiotics; neutropenic sepsis clerking documents to guide nurses/junior doctors with limited experience of such patients; and the opportunity for appropriately trained nurses to administer initial antibiotics without a doctor's prescription.

We hope that disseminating the full range of creative and practical responses to delayed door-to-needle times will promote debate as to what constitutes best practice and how best to achieve it. What is conspicuous by its absence is whether any of this has a tangible impact on patient outcomes. Without concrete data pertaining to neutropenic sepsis specifically, both the positive and negative putative effects of rapid, intravenous antibiotic therapy are at best speculative. It is conceivable, for example, that speed of fluid resuscitation is at least as important as door-to-needle time, yet there are currently no national targets for this aspect of immediate management. Other have called for a national clinical trial of best management of neutropenic sepsis [START_REF] Richardson | Improving management of neutropenic sepsis in the emergency department[END_REF][START_REF] Innes | Management of febrile neutropenia in the United Kingdon: time for a national trial?[END_REF], a recommendation which these audit findings serve only to reinforce.

A second clear gap in the evidence concerns the potential impact of delays from first symptom onset to first presentation to a healthcare provider (so-called "patient delay" as opposed to "provider delay"). Only one local audit examined this issue, finding a median patient delay of 9 h (range 1 h to 4 days). Further research is necessary to establish more definitive data for the extent of patient delays, and for the efficacy of interventions aimed at reducing them. In conclusion, the regional inconsistencies in local management of neutropenic sepsis, plus the UK-wide gulf between actual performance and current gold standards, highlight the need for further research evaluating both the feasibility and the impact on outcomes of a one hour door-to-needle time.

F

  meropenem or imipenem alone, or Tazocin plus gentamicin when specific conditions were met (for example, when the patient demonstrates clinical features of septic shock).

  Time to administration of first dose of antibiotics ("door-to-needle time") 25 of the 27 audits received (n=590) documented door-to-needle time for administration of first dose of intravenous antibiotics. Only 26% of patients received antibiotics within the target time of one hour. Median door-to-needle times (data from 16 audits) ranged from 55 min to 4 h (Fig1) with only two audits demonstrating a median delay of under 1 of over 5 h were not uncommon, the longest being 21, 22 and 24 h respectively.

  being physically unavailable or hard to locate in emergency departments/MAUs (7%)

  Table IV Reasons for delayed door-to-needle time, categorised by type 1. Lack of staff Prolonged time to: (a) nurse triage (b) assessment by junior doctor Lack of senior staff to provide second opinion on patient Delay in patient being weighed before receiving gentamicin dose 2. Lack of understanding Lack of awareness of: (a) natural evolution of course of neutropenic sepsis, particularly how quickly an apparently well patient can deteriorate (b) importance of early antibiotic administration (c) existence of protocol in ED/MAU Protocol: (a) not physically available, or hard to locate, in ED/MAU (b) too long or unwieldy to read in full Junior doctor delaying prescribing antibiotics until after: (a) full blood count is known (b) renal function is known (c) senior or haematology review Confusion about which antibiotics to use, particularly among junior doctors covering wards/MAU at night Patient waiting in ED despite showing their alert card Ambulance not provided for patient because 999 call not classified as an emergency 3. Practical problems Lack of available inpatient beds on specialist ward ED not stocking appropriate intravenous antibiotics Complex admission pathways involving 4 h stay in ED, transfer to MAU, then transfer to medical/specialist ward Patient unable to be cannulated by nurse/junior doctor 4. Communication issuesAntibiotics prescribed by doctor but not given by nurse, or until patient has been transferred to specialist ward Patient's management plan not made explicit, either verbally or in writing, so no importance given to administration of antibiotics Lack of "ownership" of patient's management plan, due to multiple team involvement (ED/MAU/specialist etc)

	Table I Table II Table III Fig 1 Fig 2
	Data included Definitions of febrile Specific instructions given in protocols which might influence outcome
	Instruction	Number (%) Temperature (⁰C)*	Total population covered Number of protocols No of protocols
	Completed questionnaires >37•5	61 (100)	n/a	4	Yes	No
	Protocols To be guided by clinical signs of sepsis, not just temperature 55 (90) > 38•0 or 2 x > 37.0	29,370,000 1 35 (71%) 14 (29%)
	Audits Not to wait for full blood count before administering antibiotics 27 (44) > 38•0 or 2 x > 37.5	627	7	26 (53%) 23 (47%)
	> 38•0 To administer first dose of empirical antibiotics within 1 hour maximum		10	26 (53%) 23 (47%)
	2 x > 38•0 To inform immediately the on-call haematology/oncology team of patient's arrival	2	19 (39%) 30 (61%)
		> 38•3 or 2 x > 38•0		5
		> 38•5 or 2 x > 38•0		20
		> 39•0 or 2 x > 38•0		4
		> 39•0 or 2 x > 38•4		1
		No formal definition provided		1
		o r P e e r R e v i e w * where 2 x readings are given, these are at least 1 hour apart F o r F o r F o r F o r F o r F o r F o r F o r P e P P P P P P P e e e e e e e e r e r e r e r e r e r e r e r R e R R R R R R R v e e e e e e e i e w v i e v i e v i e v i e v i e v i e v i e
				w w w w w w w
							11

British Journal of Haematology

Acknowledgements

We would like to thank all those who kindly provided us with data from their haematology departments, and Kate Amberley from the British Society for Haematology for her logistical support. We are also grateful to Dr Jon Salmon, Dr Patrick Medd and Dr Graham Collins for their helpful comments on the manuscript.

RC wrote the first draft of the paper and all authors contributed to subsequent drafts. RC, JW and KS extracted and analysed the data. TL and RC designed the study.