iron deficiency ( i d ) continues to be one of the most prevalent nutritional deficiencies in the world , particularly in developing countries ( world health organization ( who ) , 2003 ) . who estimated that i d occurs in about 6680% of the world 's population ( who , 2003 ) . i d has many negative effects on health , including changes in immune function , cognitive development , temperature regulation , energy metabolism and work performance ( dallman , 1986 ) . on the other hand , iron is a pro - oxidant and thus a possible risk factor for coronary heart disease . however , a recent review suggests that there is no strong association between body iron stores and coronary heart disease in adults ( zegrean , 2009 ) . the diagnosis of i d is based primarily on laboratory measurements of biochemical iron indicators . there is no full international consensus on the indicators to be used for assessing iron status , as each indicator has its own limitations because of either poor sensitivity or specificity , or because it is modified by conditions other than i d ( dallman et al . , 1993 ) . there are three general approaches to assess i d in populations . in the first one , used in studies conducted by hallberg et al . ( hallberg et al . , 1993 ) and amstrong ( amstrong , 1989 ) , i d is defined as a low level of serum ferritin ( sf ) . in the second one , used in the us in the national health and nutrition examination survey ( nhanes ) ii and iii , the multiple - criteria model is used to classify i d ; it consists of two or three abnormal values for transferrin saturation , sf and erythrocyte protoporphyrin ( cogswell et al . , 2009 ) . the main limitation of these two approaches is that inflammation leads to high ferritin values , even though iron stores are low . the third approach proposed by who ( 2004 ) and adopted by zimmermann ( 2008 ) is based on the measurements of sf and soluble transferrin receptor ( stfr ) in combination with c - reactive protein ( crp ) to eliminate false negative cases in presence of inflammation . the third approach was considered more appropriate and was therefore used in the present study . it is well known that sf concentrations reflect the size of the iron stores ( cook , 1999 ) and that low sf levels identify  iron depletion ' ( who , 2001 ) . on the other hand , tissue i d ' ( skikne et al . , 1990 ) and its values are not influenced by acute or chronic inflammatory conditions ( ferguson et al . 2003 ) using the formula : body iron ( mg / kg)=(log(stfr / sf)2.8229)/0.1207 to allow the full range of the iron status of a population to be evaluated , with negative values representing a deficit of iron with respect to the quantity required to maintain a normal haemoglobin ( hb ) concentration . adolescents are at risk of i d because of their high iron requirements , especially during the growth spurt period ( moreno , 2008 ) . in boys , increased muscular growth leads to increased demand for iron . in girls , in addition to the fast growth , the onset of menstruation leads to iron losses ( wharton and wharton , 1987 ) . as a consequence , i d occurs in adolescents also in developed countries , if their additional iron needs are not met ( dallman et al . , some studies suggested that i d can have detrimental effects on memory and learning processes in preadolescents and adolescents ( beard and connor , 2003 ) . the data available on iron status in european adolescents , are derived from studies utilising different methodologies , different indicators of iron status and varying criteria to define i d ( hercberg et al . , 2001 ) . there was , therefore , a need to investigate the situation of i d among european adolescents with a harmonised methodology . within the framework of the helena study ( healthy lifestyle in europe by nutrition in adolescents ) , funded within the 6th eu framework programme , a biochemical evaluation of iron status was performed with a cross - sectional approach . the objective of the present paper is to report the iron status of european adolescents . a detailed description of helena - cross - sectional study ( helena - css ) in terms of sampling and recruitment approaches , standardisation and harmonisation processes , data collection , analysis strategies and quality - control activities has been published elsewhere ( moreno et al . , 2008 ) . the study was approved by the research ethics committees of each country involved according to the declaration of helsinki and international conferences on harmonization for good clinical practice . written informed consent was obtained from the parents of the adolescents and the adolescents themselves ( bghin et al . , 2008 ) . briefly , subjects with an age range of 12.517.49 years were recruited in 10 cities across europe . selection of cities was based on two criteria : regional distribution and presence of an active research group assuring sufficient expertise and resources to successfully perform epidemiological studies . within the study , athens , heraklion ( greece ) , rome ( italy ) and zaragoza ( spain ) represented southern europe . ghent ( belgium ) and lille ( france ) represented western europe , whereas dortmund ( germany ) and vienna ( austria ) represented central europe . as the overall aim of the helena study was to assess the nutritional status of the adolescent population in europe , during the design phase , the variability of a large set of nutritional status indices obtained in previous studies was checked . therefore , the sample size was estimated according to the mean body mass index and variance values for each sex and age strata . for the sample size calculation , a confidence level of 95% and an error of  0.3  kg / m were assumed , leading to a sample size of 3000 adolescents . therefore , the total expected subsample was 1000 adolescents ( 100 subjects for each city ) . the size of subsample was chosen , so as to assess the immunological parameters with sufficient precision . such parameters were those with the highest variability within the blood measurements that were included in the study as discussed by moreno et al . the selection procedure for schools and classes including the selection of a subset of classes for blood sampling has been applied centrally by one partner ( ghent ) for all study centres . the blood sampling procedure and sample logistics have been described in detail elsewhere ( gonzlez - gross et al . , 2008 ) . briefly , fasting blood samples were collected by venipuncture at school between 0800 and 1000 hours in the morning . whole - blood samples for the red blood parameters ( hb , red blood cell , mean corpuscular volume ( mcv ) , mean corpuscular haemoglobin ( mch ) , mean corpuscular haemoglobin concentration and red cell distribution width ) were sent directly to the local laboratory of each country to be analysed . samples for iron status analyses were clotted at room temperature for at least 30  min , followed by centrifuged at 3500  r.p.m . for 10  min . serum samples were shipped to the central laboratory in bonn university ( iel , germany ) , department of nutrition and food science , to be stored at 80  c before being shipped to italy in may 2007 , within 6 months of the collection . the aliquots of serum samples were analysed in the human nutrition laboratory of the national research institute on food and nutrition ( inran ) . sf , stfr and crp were measured by using an enzyme linked immunosorbent assay ( erhardt et al . , 2004 ) . a commercially available control sample from bio - rad liquichek immunology control level 3 ( bio - rad , milan , italy ) was used to obtain a calibration curve on each plate . a quality - control serum was produced in house by pooling human serum from four healthy volunteers to monitor accuracy and precision of the determinations . the coefficient of variation from 10 replicates of the quality - control serum pool was 5.3 , 6.4 and 12.4% for sf , stfr and crp , respectively . additionally , the interassay coefficient of variation of the pool samples analysed in a total of 32 different analytical runs ( four replicates ) was 10.8 , 14.8 and 23.4% for sf , stfr and crp , respectively . inran laboratory is involved in an external quality assurance program of cdc vital - eqa for sf , stfr and crp analyses ( haynes et al . , 2008 ) . in spring 2007 , the measures of iron indicators of this study were within the acceptable range , except for crp that was just below the inferior limit ( 2.42  mg / l versus an acceptable range of 2.705.38  mg / l ) and stfr that was at the superior limit ( 7.3  mg / l versus an acceptable range of 1.7 and 7.3 anaemia was defined as hb concentration below the cutoff set by who ( who , 2004 ) , 12.0  g / dl for girls aged 12 years and above , and for boys aged 1214 ; and 13.0  g / dl for boys aged 15 years or above . abnormal values for iron indicators were defined as concentration < 15  g / l for low sf ( who , 2004 ) , > 200  g / l and > 150  g / l ( boys and girls , respectively ) for high sf ( who , 2004 ; severe risk of iron overload ) and > 8.5  mg / l for high stfr ( cook et al . , 1993 ) . a participant was considered to have active inflammation , if the crp concentration exceeded 5  mg / l ( woodruff et al . , 2006 ) . total body iron was estimated with the use of the formula : body iron ( mg / kg)=(log(stfr / sf)2.8229)/0.1207 ( cook et al . , 2003 ) . wallis test was used for comparing haematological parameters among iron sufficiency , iron depletion and i d . the student 's t - test was used to evaluate the significance of differences between mean values of iron indicators between genders . iron status indicators , such as stfr and sf , were transformed to logarithms before performing statistical analyses . the results in the tables are shown using the original units without any log transformation . as already performed by ortega et al . ( ortega et al . , 2010 ) , all the analyses conducted on the helena data were adjusted by a weighing factor to balance the sample according to the age and sex distribution of the theoretical sample to guarantee representation of each of the stratified groups . the counts of subjects ( n ) are reported in the tables as unweighted values . all the statistical analyses were performed using the spss statistical software package for microsoft windows ( version 12.00 , spss inc . , chicago , il , usa ) . a detailed description of helena - cross - sectional study ( helena - css ) in terms of sampling and recruitment approaches , standardisation and harmonisation processes , data collection , analysis strategies and quality - control activities has been published elsewhere ( moreno et al . , 2008 ) . the study was approved by the research ethics committees of each country involved according to the declaration of helsinki and international conferences on harmonization for good clinical practice . written informed consent was obtained from the parents of the adolescents and the adolescents themselves ( bghin et al . , 2008 ) . briefly , subjects with an age range of 12.517.49 years were recruited in 10 cities across europe . selection of cities was based on two criteria : regional distribution and presence of an active research group assuring sufficient expertise and resources to successfully perform epidemiological studies . within the study , athens , heraklion ( greece ) , rome ( italy ) and zaragoza ( spain ) represented southern europe . ghent ( belgium ) and lille ( france ) represented western europe , whereas dortmund ( germany ) and vienna ( austria ) represented central europe . as the overall aim of the helena study was to assess the nutritional status of the adolescent population in europe , during the design phase , the variability of a large set of nutritional status indices obtained in previous studies was checked . therefore , the sample size was estimated according to the mean body mass index and variance values for each sex and age strata . for the sample size calculation , a confidence level of 95% and an error of  0.3  kg / m were assumed , leading to a sample size of 3000 adolescents . therefore , the total expected subsample was 1000 adolescents ( 100 subjects for each city ) . the size of subsample was chosen , so as to assess the immunological parameters with sufficient precision . such parameters were those with the highest variability within the blood measurements that were included in the study as discussed by moreno et al . the selection procedure for schools and classes including the selection of a subset of classes for blood sampling has been applied centrally by one partner ( ghent ) for all study centres . the blood sampling procedure and sample logistics have been described in detail elsewhere ( gonzlez - gross et al . , 2008 ) . briefly , fasting blood samples were collected by venipuncture at school between 0800 and 1000 hours in the morning . whole - blood samples for the red blood parameters ( hb , red blood cell , mean corpuscular volume ( mcv ) , mean corpuscular haemoglobin ( mch ) , mean corpuscular haemoglobin concentration and red cell distribution width ) were sent directly to the local laboratory of each country to be analysed . samples for iron status analyses were clotted at room temperature for at least 30  min , followed by centrifuged at 3500  r.p.m . for 10  min . serum samples were shipped to the central laboratory in bonn university ( iel , germany ) , department of nutrition and food science , to be stored at 80  c before being shipped to italy in may 2007 , within 6 months of the collection . the aliquots of serum samples were analysed in the human nutrition laboratory of the national research institute on food and nutrition ( inran ) . sf , stfr and crp were measured by using an enzyme linked immunosorbent assay ( erhardt et al . , 2004 ) . a commercially available control sample from bio - rad liquichek immunology control level 3 ( bio - rad , milan , italy ) was used to obtain a calibration curve on each plate . a quality - control serum was produced in house by pooling human serum from four healthy volunteers to monitor accuracy and precision of the determinations . the coefficient of variation from 10 replicates of the quality - control serum pool was 5.3 , 6.4 and 12.4% for sf , stfr and crp , respectively . additionally , the interassay coefficient of variation of the pool samples analysed in a total of 32 different analytical runs ( four replicates ) was 10.8 , 14.8 and 23.4% for sf , stfr and crp , respectively . inran laboratory is involved in an external quality assurance program of cdc vital - eqa for sf , stfr and crp analyses ( haynes et al . , , the measures of iron indicators of this study were within the acceptable range , except for crp that was just below the inferior limit ( 2.42 mg / l versus an acceptable range of 2.705.38  mg / l ) and stfr that was at the superior limit ( 7.3  mg / l versus an acceptable range of 1.7 and 7.3 anaemia was defined as hb concentration below the cutoff set by who ( who , 2004 ) , 12.0  g / dl for girls aged 12 years and above , and for boys aged 1214 ; and 13.0  g / dl for boys aged 15 years or above . l for low sf ( who , 2004 ) , > 200  g / l and > 150  g / l ( boys and girls , respectively ) for high sf ( who , 2004 ; severe risk of iron overload ) and > 8.5  mg / l for high stfr ( cook et al . , 1993 ) . a participant was considered to have active inflammation , if the crp concentration exceeded 5  mg / l ( woodruff et al . , 2006 ) . total body iron was estimated with the use of the formula : body iron ( mg / kg)=(log(stfr / sf)2.8229)/0.1207 ( cook et al . , 2003 ) . the kruskal  wallis test was used for comparing haematological parameters among iron sufficiency , iron depletion and i d . the student 's t - test was used to evaluate the significance of differences between mean values of iron indicators between genders . iron status indicators , such as stfr and sf , were transformed to logarithms before performing statistical analyses . the results in the tables are shown using the original units without any log transformation . as already performed by ortega et al . ( ortega et al . , 2010 ) , all the analyses conducted on the helena data were adjusted by a weighing factor to balance the sample according to the age and sex distribution of the theoretical sample to guarantee representation of each of the stratified groups . the counts of subjects ( n ) are reported in the tables as unweighted values . all the statistical analyses were performed using the spss statistical software package for microsoft windows ( version 12.00 , spss inc . , chicago , il , usa ) . a total of 102 subjects were excluded because at least one of the iron indicators ( sf , n=80 ; stfr , n=84 ) or hb ( n=16 ) was missing . the final sample of 987 adolescents included 465 boys and 522 girls , that is , 90.7% of the adolescents who participated in the blood sampling . a total of 47 subjects had high levels of crp ( 27 boys and 20 girls ) and were excluded . therefore , the present study of iron status was performed on a total of 940 adolescents ( 438 boys and 502 girls ) , with no indication of the presence of inflammation . the mean age ( standard deviation ) of adolescents was 14.7 ( 1.2 ) years both in boys and in girls , and the mean body mass index was 21.2 ( 3.8 ) and 21.1 ( 3.3 ) kg / m , respectively , for boys and girls . the reported age of menarche in the present study ranged from 9 to 16 years of age . a total of 58 ( 12% ) girls reported not to have experienced menarche at the time of the study . in all , 39 and 43% of the boys and girls , respectively , declared to have smoked cigarettes at least once . mean , median and percentiles ( 2575th ) values of red blood parameters , sf and stfr are summarised in table 2 . percentage of iron sufficiency , abnormal values of iron indicators , anaemia , iron depletion , i d and iron deficiency anaemia ( ida ) are presented in table 3 . in boys , the mean ( standard deviation ) of hb was 14.7 ( 1.2 ) g / dl ( 14.3 ( 1.0 ) g / dl aged 12.514.99 years and 15.2 ( 1.2 ) g / dl aged 15 years or above ) . the mean values of sf suggest a gender - related significant difference ( p<0.05 ) with a lower mean sf value in girls ( 27.9  g / l ) than in boys ( 36.6  g / l ) . the percentage of low sf values was also higher in girls ( 26.3% ) compared with boys ( 17.9% ) ( p<0.01 ) ( table 3 ) . l for boys and > 150  g / l for girls ) , indicating no risk of iron overload ( who , 2004 ) in the study sample . a total of 23 subjects had high values ( > 8.5  mg / l ) of stfr ( that could have indicated i d ) but with normal values of sf ( indicating no iron depletion ) , and could therefore not be categorised in one of the iron status classifications ( table 1 ) . the percentage of iron depletion was 17.6% , significantly higher in girls ( 21.0% ) than in boys ( 13.8% ) ( p<0.05 ) ( table 3 ) . the overall prevalence of anaemia was 4.4% and it ranged from 1.1% ( stockholm ) to 10.1% ( heraklion ) among cities . intermediate prevalences were 1.4% for zaragoza , 3.2% for pecs , 3.6% for dortmund , 4.0% for rome , 4.6% for ghent , 5.5% for lille , 5.6% for vienna and 5.7% for athens . the overall prevalence of anaemia without i d was 3.1% ( 4.6% in girls and 1.4% in boys ) and ranged from 0% ( stockholm and zaragoza ) to 7.6% ( heraklion ) . intermediate prevalences were 1.8% for dortmund , 1.9% for vienna , 3.0% for rome , 3.2% for pecs , 4.1% for lille and 4.6% for ghent . percentage of iron depletion ranged from 9.6% ( zaragoza ) to 23.0% ( pecs ) . percentage of i d ranged from 0% ( lille ) to 11.3% ( athens ) . percentage of ida ranged from 0% ( athens , gent , and pecs ) to 2.8 % ( vienna ) . among adolescents with anaemia ( n=40 ) , 27.5% ( n=11 ) were classified as ida , with abnormal sf and stfr values , whereas 24.3% ( n=10 ) had abnormal sf values but with normal stfr values ( iron depletion without i d ) and 47.5% ( n=19 ) had anaemia without i d , with normal sf and stfr values . log(sf ) and log(stfr ) were significantly correlated with the red cell parameters ( mcv , mch , mean corpuscular haemoglobin concentration and red cell distribution width ) ( table 4 ) . in the total sample , a significant correlation was observed between log(sf ) and hb concentration ( r=0.36 , p<0.01 ) , and between log(sf ) and log(stfr ) ( r=0.33 , p<0.01 ) . for log(stfr ) , a significant correlation was observed with log(sf ) , mch ( r=  0.30 , p<0.01 ) and mcv ( r=0.26 , p<0.01 ) no significant correlation was observed between red blood cell and iron indicators ( log(sf ) and log(stfr ) ; r=0.13 and r=0.07 , respectively ) . the mean of red cell parameters in the different classification of iron status is reported in table 5 . statistically significant differences between iron sufficiency , iron depletion and i d were observed for red cell parameters for boys and girls . there were significant differences ( p<0.001 ) for hb , mcv , mch , mean corpuscular haemoglobin concentration and red cell distribution width between iron sufficiency group and the iron depletion and i d groups . no significant difference was observed between iron sufficiency group and i d group for any of the red cell parameters considered . mean ( standard deviation ) body iron was significantly higher ( p<0.001 ; student 's t - test ) in boys ( 4.4 ( 3.2 ) mg / kg ) than in girls ( 3.3 ( 4.1 ) mg / kg ) . girls showed a light shift of the body iron distribution curve to the left in comparison with boys in which higher positive values were observed . the reference line indicates the cutoff point for tissue i d ( body iron < 0  mg / kg ) . the percentage of negative values was significantly higher ( p<0.001 ;  ) in girls ( 16.5% ) with respect to boys ( 8.3% ) . figure 2 shows that the linear relationship between the body iron and the age of the adolescents is not very strong . the body iron increased slightly with age ( r=0.28 ) but this trend did not reach statistical significance . on the other hand , it tended to be stable ( r=0.025 ) in girls . a total of 102 subjects were excluded because at least one of the iron indicators ( sf , n=80 ; stfr , n=84 ) or hb ( n=16 ) was missing . the final sample of 987 adolescents included 465 boys and 522 girls , that is , 90.7% of the adolescents who participated in the blood sampling . a total of 47 subjects had high levels of crp ( 27 boys and 20 girls ) and were excluded . therefore , the present study of iron status was performed on a total of 940 adolescents ( 438 boys and 502 girls ) , with no indication of the presence of inflammation . the mean age ( standard deviation ) of adolescents was 14.7 ( 1.2 ) years both in boys and in girls , and the mean body mass index was 21.2 ( 3.8 ) and 21.1 ( 3.3 ) kg / m , respectively , for boys and girls . the reported age of menarche in the present study ranged from 9 to 16 years of age . a total of 58 ( 12% ) girls reported not to have experienced menarche at the time of the study . in all , 39 and 43% of the boys and girls , respectively , declared to have smoked cigarettes at least once . mean , median and percentiles ( 2575th ) values of red blood parameters , sf and stfr are summarised in table 2 . percentage of iron sufficiency , abnormal values of iron indicators , anaemia , iron depletion , i d and iron deficiency anaemia ( ida ) are presented in table 3 . in boys , the mean ( standard deviation ) of hb was 14.7 ( 1.2 ) g / dl ( 14.3 ( 1.0 ) g / dl aged 12.514.99 years and 15.2 ( 1.2 ) g / dl aged 15 years or above ) . the mean values of sf suggest a gender - related significant difference ( p<0.05 ) with a lower mean sf value in girls ( 27.9  g / l ) than in boys ( 36.6  g / l ) . the percentage of low sf values was also higher in girls ( 26.3% ) compared with boys ( 17.9% ) ( p<0.01 ) ( table 3 ) . l for boys and > 150  g / l for girls ) , indicating no risk of iron overload ( who , 2004 ) in the study sample . a total of 23 subjects had high values ( > 8.5  mg / l ) of stfr ( that could have indicated i d ) but with normal values of sf ( indicating no iron depletion ) , and could therefore not be categorised in one of the iron status classifications ( table 1 ) . the percentage of iron depletion was 17.6% , significantly higher in girls ( 21.0% ) than in boys ( 13.8% ) ( p<0.05 ) ( table 3 ) . the overall prevalence of anaemia was 4.4% and it ranged from 1.1% ( stockholm ) to 10.1% ( heraklion ) among cities . intermediate prevalences were 1.4% for zaragoza , 3.2% for pecs , 3.6% for dortmund , 4.0% for rome , 4.6% for ghent , 5.5% for lille , 5.6% for vienna and 5.7% for athens . the overall prevalence of anaemia without i d was 3.1% ( 4.6% in girls and 1.4% in boys ) and ranged from 0% ( stockholm and zaragoza ) to 7.6% ( heraklion ) . intermediate prevalences were 1.8% for dortmund , 1.9% for vienna , 3.0% for rome , 3.2% for pecs , 4.1% for lille and 4.6% for ghent . percentage of iron depletion ranged from 9.6% ( zaragoza ) to 23.0% ( pecs ) . percentage of i d ranged from 0% ( lille ) to 11.3% ( athens ) . percentage of ida ranged from 0% ( athens , gent , and pecs ) to 2.8 % ( vienna ) . among adolescents with anaemia ( n=40 ) , 27.5% ( n=11 ) were classified as ida , with abnormal sf and stfr values , whereas 24.3% ( n=10 ) had abnormal sf values but with normal stfr values ( iron depletion without i d ) and 47.5% ( n=19 ) had anaemia without i d , with normal sf and stfr values . log(sf ) and log(stfr ) were significantly correlated with the red cell parameters ( mcv , mch , mean corpuscular haemoglobin concentration and red cell distribution width ) ( table 4 ) . in the total sample , a significant correlation was observed between log(sf ) and hb concentration ( r=0.36 , p<0.01 ) , and between log(sf ) and log(stfr ) ( r=0.33 , p<0.01 ) . for log(stfr ) , a significant correlation was observed with log(sf ) , mch ( r=  0.30 , p<0.01 ) and mcv ( r=0.26 , p<0.01 ) no significant correlation was observed between red blood cell and iron indicators ( log(sf ) and log(stfr ) ; r=0.13 and r=0.07 , respectively ) . the mean of red cell parameters in the different classification of iron status is reported in table 5 . statistically significant differences between iron sufficiency , iron depletion and i d were observed for red cell parameters for boys and girls . there were significant differences ( p<0.001 ) for hb , mcv , mch , mean corpuscular haemoglobin concentration and red cell distribution width between iron sufficiency group and the iron depletion and i d groups . no significant difference was observed between iron sufficiency group and i d group for any of the red cell parameters considered . mean ( standard deviation ) body iron was significantly higher ( p<0.001 ; student 's t - test ) in boys ( 4.4 ( 3.2 ) mg / kg ) than in girls ( 3.3 ( 4.1 ) mg / kg ) . girls showed a light shift of the body iron distribution curve to the left in comparison with boys in which higher positive values were observed . the reference line indicates the cutoff point for tissue i d ( body iron < 0  mg / kg ) . the percentage of negative values was significantly higher ( p<0.001 ;  ) in girls ( 16.5% ) with respect to boys ( 8.3% ) . figure 2 shows that the linear relationship between the body iron and the age of the adolescents is not very strong . the body iron increased slightly with age ( r=0.28 ) but this trend did not reach statistical significance . on the other hand , it tended to be stable ( r=0.025 ) in girls . the present study is the first report evaluating iron status in adolescents across multiple european countries . as discussed by moreno et al . ( 2008 ) , the selected cities were equivalent and comparable between countries but the samples were representative for the cities and not for the countries . therefore , the helena study does not allow for statistical comparison of differences in iron status between regions . however , the prevalence of iron status stages according to geographical location of the cities adopted for this study are provided underneath for descriptive purpose . in the present study , the overall proportion of iron depletion was 17.6% it was significantly higher in girls ( 21.0% ) than in boys ( 13.8% ) . the prevalence of iron depletion by geographical location was : 23% in eastern europe ( pecs ) , 19% in northern europe ( stockholm ) , 18% in western europe ( 17% in ghent and 19% in lille ) , 17% in central europe ( 16% in dortmund and 19% in vienna ) and 15% in southern europe ( 14% in athens , 17% in heraklion , 19% in rome and 10% in zaragoza ) . these data are in the range of findings from a recent review on iron status in european adolescents ( hercberg et al . , 2001 ) where the highest prevalence of iron depletion was observed in girls in ireland ( 43% ) and in boys in denmark ( 16% ) , whereas the lowest prevalence was observed in girls in finland ( 4.7% ) and in boys in france where no cases were detected . for i d , in the present study , there was a tendency for a higher percentage in girls ( 5.4% ) with respect to boys ( 3.9% ) but the difference was not significant . the percentage of i d in girls in our study was slightly lower than the 8.7% prevalence observed in girls aged 1216 years in the us ( halterman et al . , 2001 ) but in line with the 6% prevalence observed in girls aged 1319 years in canada ( deegan et al . , 2005 ) both the prevalences observed in our study and in the us are below the cutoff established by who to determine  no prevalence ' of i d , that is , 10% ( who , 2004 ) . both are also far lower than that observed in other parts of the world as in nepal , with an i d prevalence of 56% observed in adolescents girls ( woodruff et al . , 2006 ) . the slightly lower prevalence in our study may , in part , result from a different approach to define i d . the impact of i d on the cognitive functioning of adolescent girls who are at the highest risk of i d requires major clarification . in fact , there is only limited evidence that iron supplementation may improve attention , concentration and intelligent quotient ( falkingham et al . , 2010 ) . the prevalence of i d by geographical location was : 6% in both northern europe ( stockholm ) and southern europe ( 11% in athens , 6% in heraklion , 3% in rome and zaragoza ) plus eastern europe ( pecs ) , 5% in central europe ( 3% in dortmund and 7% in vienna ) and 2% in western europe ( no cases in ghent and 4% in lille ) . no comparable data are available in the literature in relation to i d in adolescents in the european context for comparison with the results of the present study . the percentage of ida was 1.3% ( 0.5% in boys and 2.0% in girls ) , with a ( non - significantly ) higher value in girls . the prevalence of ida by geographical location was : 2% in central europe ( 2% in dortmund and 3% in vienna ) , 1% in both northern europe ( stockholm ) and southern europe ( no cases in athens , 2% in heraklion , 1% in both rome and zaragoza ) , 0.6% in western europe ( no cases in ghent and 1% in lille ) and no cases in eastern europe ( pecs ) . these data are similar to the estimates of prevalence in adolescents in canada ( deegan et al . , 2005 ) , the united states ( looker et al . , 2002 ) and sweden ( samulson et al . , 1996 ) , ranging from 0 to 3% . in the present study , the proportion of anaemia was 4.4% , consistent with the estimates of the worldwide prevalence of anaemia during adolescence : 27% in developing countries and 6% in developed countries ( kara et al . the observed prevalence of anaemia not due to i d ( 3.1% ) is probably overestimated because of unavoidable misclassifications that are inherent to the definition of limits . in fact , the limits set to define anaemia are based on 95% reference ranges , and therefore it is assumed that 2.5% of healthy individuals would be classified as anaemic . on the other hand , a proportion of anaemia without i d may be originated from genetic hb disorders , as subjects with such disorders were not excluded . in our study , the highest prevalence of anaemia without i d among cities was observed in two cities of greece ( 7.6% in heraklion and 6.3% in athens ) . greece is known to be heavily affected by haemoglobinopathies ( thalassaemia and sickle - cell disease ) . ( 1995 ) , the highest percentage of carriers of thalassaemia and sickle - cell disorders is observed in greece ( 9.0% ) . in our study , no single case of iron overload was identified through sf , a stored body iron marker . results of the present study on correlations between iron status parameters are all plausible . sf and stfr that were chosen in this study as iron indicators showed a correlation with red cell parameters . the observed correlation coefficients between log(stfr ) and red cell parameters ( mcv and mch ) are not surprising , as stfr is a measure of i d erytropoiesis ( metzgeroth et al . , 2005 ) as mcv and mch . the present data are in general agreement with the result of a previous study showing a correlation between stfr and red cell parameters in iron - deficient adolescents ( choi , 2003 ) . these findings are confirmed , as the concentrations of the mean of red cell parameters decreased gradually ( except for red cell distribution width that increased ) with increasingly negative iron balance , as i d appeared ( stfr > 8.5  mg / l ) ( table 5 ) . the results of the present study document a positive correlation between log(sf ) level and hb concentration ( r=0.39 , p<0.01 ; table 3 ) comparable to those observed in other studies among children : r=0.24 ( jeremiah et al . , 2007 ) and r=0.26 ( ayoya et al . , 2010 ) . the low correlation between these two indicators is because of the fact that they are related to differing stages of i d . the correlation between the indicators of iron status and red cell parameters may be considered as a confirmation of the reliability of these indicators ( who , 2004 ; zimmermann , 2008 ) in the assessment of iron status in adolescents . the correlation of sf with hb and the higher percentage of iron depletion in girls with respect to boys suggest that the risk of developing i d anaemia is higher in girls . in addition , the calculated measure of body iron was lower in girls than in boys . the distribution of body iron stores by age ( figure 2 ) suggests that in boys , body iron stores may have slightly increased during and after their rapid growth period , whereas in girls , iron stores remain low during the whole pubertal period . one possible explanation for the lower iron status in girls , besides growth , is the loss of iron with menstruation . in previous studies , it was observed that the growth spurt associated with menstrual status , blood loss and a low iron intake may have adverse effects on iron stores in adolescents girls ( ilich - ernst et al . our study confirms sf and stfr as biochemical indicators that could be used in the assessment of iron status in adolescents , a problematic group of population , for the period of rapid growth . on the basis of who guidelines ( 2004 ) for the interpretation of the iron status data at population level , the results indicate that , in the european context , i d is not prevalent neither in boys nor in girls , and that iron depletion is prevalent in girls but not in boys . adolescent girls therefore constitute a group at risk of i d , and specific attention should be given to them during adolescence to ensure that their dietary intake of iron is adequate to their requirements . co - ordinator : luis a moreno core group members : luis a moreno , frderic gottrand , stefaan de henauw , marcela gonzlez - gross , chantal gilbert . steering committee : anthony kafatos ( president ) , luis a moreno , christian libersa , stefaan de henauw , jackie schez , frderic gottrand , mathilde kesting , michael sjostrom , dnes molnr , marcela gonzlez - gross , jean dallongeville , chantal gilbert , gunnar hall , lea maes , luca scalfi . project manager : pilar melndez 
 universidad de zaragoza ( spain ) : luis a moreno , jess fleta , jos a casajs , gerardo rodrguez , concepcin toms , mara i mesana , germn vicente - rodrguez , adoracin villarroya , carlos m gil , ignacio ara , juan revenga , carmen lachen , juan fernndez alvira , gloria bueno , aurora lzaro , olga bueno , juan f len , jess m garagorri , manuel bueno , juan pablo rey lpez , iris iglesia , paula velasco , silvia bel.consejo superior de investigaciones cientficas ( spain ) : ascensin marcos , julia wrnberg , esther nova , sonia gmez , esperanza ligia daz , javier romeo , ana veses , mari angeles puertollano , beln zapatera , tamara pozo.universit de lille 2 ( france ) : laurent bghin , christian libersa , frdric gottrand , catalina iliescu , juliana von berlepsch.research institute of child nutrition dortmund , rheinische friedrich - wilhelms - universitt bonn ( germany ) : mathilde kersting , wolfgang sichert - hellert , ellen koeppen.pcsi tudomnyegyetem ( university of pcs ) ( hungary ) : dnes molnar , eva erhardt , katalin csernus , katalin trk , szilvia bokor , mrs angster , enik nagy , orsolya kovcs , judit repsy.university of crete school of medicine ( greece ) : anthony kafatos , caroline codrington , mara plada , angeliki papadaki , katerina sarri , anna viskadourou , christos hatzis , michael kiriakakis , george tsibinos , constantine vardavas manolis sbokos , eva protoyeraki , maria fasoulaki.institut fr ernhrungs- und lebensmittelwissenschaften  ernhrungphysiologie . rheinische friedrich - wilhelms universitt ( germany ) : peter stehle , klaus pietrzik , marcela gonzlez - gross , christina breidenassel , andre spinneker , jasmin al - tahan , miriam segoviano , anke berchtold , christine bierschbach , erika blatzheim , adelheid schuch , petra pickert.university of granada ( spain ) : manuel j castillo , ngel gutirrez , francisco b ortega , jonatan r ruiz , enrique g artero , vanesa espaa - romero , david jimnez - pavn , palma chilln.istituto nazionale di ricerca per gli alimenti e la nutrizione ( italy ) : davide arcella , elena azzini , emma barrison , noemi bevilacqua , pasquale buonocore , giovina catasta , laura censi , donatella ciarapica , paola d'acapito , marika ferrari , myriam galfo , cinzia le donne , catherine leclercq , giuseppe maiani , beatrice mauro , lorenza mistura , antonella , pasquali , raffaela piccinelli , angela polito , raffaella spada , stefania sette , maria zaccaria.university of napoli  federico ii ' dept of food science ( italy ) : luca scalfi , paola vitaglione , concetta montagnese.ghent university ( belgium ) : ilse de bourdeaudhuij , stefaan de henauw , tineke de vriendt , lea maes , christophe matthys , carine vereecken , mieke de maeyer , charlene ottevaere.medical university of vienna ( austria ) : kurt widhalm , katharina phillipp , sabine dietrich.harokopio university ( greece ) : yannis manios , eva grammatikaki , zoi bouloubasi , tina louisa cook , sofia eleutheriou , orsalia consta , george moschonis , ioanna katsaroli , george kraniou , stalo papoutsou , despoina keke , ioanna petraki , elena bellou , sofia tanagra , kostalenia kallianoti , dionysia argyropoulou , katerina kondaki , stamatoula tsikrika , christos karaiskos.institut pasteur de lille ( france ) : jean dallongeville , aline meirhaeghe.karolinska institutet ( sweden ) : michael sjstrom , patrick bergman , mara hagstrmer , lena hallstrm , mrten hallberg , eric poortvliet , julia wrnberg , nico rizzo , linda beckman , anita hurtig wennlf , emma patterson , lydia kwak , lars cernerud , per tillgren , stefaan srensen.asociacin de investigacin de la industria agroalimentaria ( spain ) : jackie snchez - molero , elena pic , maite navarro , blanca viadel , jos enrique carreres , gema merino , rosa sanjun , mara lorente , mara jos snchez , sara castell.campden and chorleywood food research association ( united kingdom ) : chantal gilbert , sarah thomas , elaine allchurch , peter burguess.sikinstitutet foer livsmedel och bioteknik ( sweden ) : gunnar hall , annika astrom , anna sverkn , agneta broberg.meurice recherche and development asbl ( belgium ) : annick masson , claire lehoux , pascal brabant , philippe pate , laurence fontaine.campden and chorleywood food development institute ( hungary ) : andras sebok , tunde kuti , adrienn hegyi.productos aditivos sa ( spain ) : cristina maldonado , ana llorente.crnicas serrano sl ( spain ) : emilio garca.cederroth international ab ( sweden ) : holger von fircks , marianne lilja hallberg , maria messerer.lantmnnen food r and d ( sweden ) : mats larsson , helena fredriksson , viola adamsson , ingmar brjesson.european food information council ( belgium ) : laura fernndez , laura smillie , josephine wills.universidad politcnica de madrid ( spain ) : marcela gonzlez - gross , agustn melndez , pedro j. benito , javier caldern , david jimnez - pavn , jara valtuea , paloma navarro , alejandro urzanqui , ulrike albers , raquel pedrero , juan jos gmez lorente . universidad de zaragoza ( spain ) : luis a moreno , jess fleta , jos a casajs , gerardo rodrguez , concepcin toms , mara i mesana , germn vicente - rodrguez , adoracin villarroya , carlos m gil , ignacio ara , juan revenga , carmen lachen , juan fernndez alvira , gloria bueno , aurora lzaro , olga bueno , juan f len , jess m garagorri , manuel bueno , juan pablo rey lpez , iris iglesia , paula velasco , silvia bel . consejo superior de investigaciones cientficas ( spain ) : ascensin marcos , julia wrnberg , esther nova , sonia gmez , esperanza ligia daz , javier romeo , ana veses , mari angeles puertollano , beln zapatera , tamara pozo . universit de lille 2 ( france ) : laurent bghin , christian libersa , frdric gottrand , catalina iliescu , juliana von berlepsch . research institute of child nutrition dortmund , rheinische friedrich - wilhelms - universitt bonn ( germany ) : mathilde kersting , wolfgang sichert - hellert , ellen koeppen . pcsi tudomnyegyetem ( university of pcs ) ( hungary ) : dnes molnar , eva erhardt , katalin csernus , katalin trk , szilvia bokor , mrs angster , enik nagy , orsolya kovcs , judit repsy . university of crete school of medicine ( greece ) : anthony kafatos , caroline codrington , mara plada , angeliki papadaki , katerina sarri , anna viskadourou , christos hatzis , michael kiriakakis , george tsibinos , constantine vardavas manolis sbokos , eva protoyeraki , maria fasoulaki . rheinische friedrich - wilhelms universitt ( germany ) : peter stehle , klaus pietrzik , marcela gonzlez - gross , christina breidenassel , andre spinneker , jasmin al - tahan , miriam segoviano , anke berchtold , christine bierschbach , erika blatzheim , adelheid schuch , petra pickert . university of granada ( spain ) : manuel j castillo , ngel gutirrez , francisco b ortega , jonatan r ruiz , enrique g artero , vanesa espaa - romero , david jimnez - pavn , palma chilln . istituto nazionale di ricerca per gli alimenti e la nutrizione ( italy ) : davide arcella , elena azzini , emma barrison , noemi bevilacqua , pasquale buonocore , giovina catasta , laura censi , donatella ciarapica , paola d'acapito , marika ferrari , myriam galfo , cinzia le donne , catherine leclercq , giuseppe maiani , beatrice mauro , lorenza mistura , antonella , pasquali , raffaela piccinelli , angela polito , raffaella spada , stefania sette , maria zaccaria . university of napoli  federico ii ' dept of food science ( italy ) : luca scalfi , paola vitaglione , concetta montagnese . ghent university ( belgium ) : ilse de bourdeaudhuij , stefaan de henauw , tineke de vriendt , lea maes , christophe matthys , carine vereecken , mieke de maeyer , charlene ottevaere . medical university of vienna ( austria ) : kurt widhalm , katharina phillipp , sabine dietrich . harokopio university ( greece ) : yannis manios , eva grammatikaki , zoi bouloubasi , tina louisa cook , sofia eleutheriou , orsalia consta , george moschonis , ioanna katsaroli , george kraniou , stalo papoutsou , despoina keke , ioanna petraki , elena bellou , sofia tanagra , kostalenia kallianoti , dionysia argyropoulou , katerina kondaki , stamatoula tsikrika , christos karaiskos . karolinska institutet ( sweden ) : michael sjstrom , patrick bergman , mara hagstrmer , lena hallstrm , mrten hallberg , eric poortvliet , julia wrnberg , nico rizzo , linda beckman , anita hurtig wennlf , emma patterson , lydia kwak , lars cernerud , per tillgren , stefaan srensen . asociacin de investigacin de la industria agroalimentaria ( spain ) : jackie snchez - molero , elena pic , maite navarro , blanca viadel , jos enrique carreres , gema merino , rosa sanjun , mara lorente , mara jos snchez , sara castell . campden and chorleywood food research association ( united kingdom ) : chantal gilbert , sarah thomas , elaine allchurch , peter burguess . institutet foer livsmedel och bioteknik ( sweden ) : gunnar hall , annika astrom , anna sverkn , agneta broberg . meurice recherche and development asbl ( belgium ) : annick masson , claire lehoux , pascal brabant , philippe pate , laurence fontaine . campden and chorleywood food development institute ( hungary ) : andras sebok , tunde kuti , adrienn hegyi . cederroth international ab ( sweden ) : holger von fircks , marianne lilja hallberg , maria messerer . lantmnnen food r and d ( sweden ) : mats larsson , helena fredriksson , viola adamsson , ingmar brjesson . european food information council ( belgium ) : laura fernndez , laura smillie , josephine wills . universidad politcnica de madrid ( spain ) : marcela gonzlez - gross , agustn melndez , pedro j. benito , javier caldern , david jimnez - pavn , jara valtuea , paloma navarro , alejandro urzanqui , ulrike albers , raquel pedrero , juan jos gmez lorente .
background / objectives : to assess the iron status among european adolescents through selected biochemical parameters in a cross - sectional study performed in 10 european cities.subjects/methods:iron status was defined utilising biochemical indicators . 
 iron depletion was defined as low serum ferritin ( sf<15  g 
 / l ) . 
 iron deficiency ( i d ) was defined as high - soluble transferrin receptor ( stfr>8.5  mg / l ) plus iron depletion . 
 iron deficiency anaemia ( ida ) was defined as i d with haemoglobin ( hb ) below the who cutoff for age and sex : 12.0  g / dl for girls and for boys aged 12.514.99 years and 13.0  g / dl for boys aged 15 years . 
 enzyme linked immunosorbent assay was used as analytical method for sf , stfr and c - reactive protein ( crp ) . 
 subjects with indication of inflammation ( crp > 5 
 mg / l ) were excluded from the analyses . 
 a total of 940 adolescents aged 12.517.49 years ( 438 boys and 502 girls ) were involved.results:the percentage of iron depletion was 17.6% , significantly higher in girls ( 21.0% ) compared with boys ( 13.8% ) . 
 the overall percentage of i d and ida was 4.7 and 1.3% , respectively , with no significant differences between boys and girls . a correlation was observed between log ( sf ) and hb ( r=0.36 , p<0.01 ) , and between log ( stfr ) and mean corpuscular haemoglobin ( r=0.30 , p<0.01 ) . 
 iron body stores were estimated on the basis of log ( stfr / sf ) . 
 a higher percentage of negative values of body iron was recorded in girls ( 16.5% ) with respect to boys ( 8.3% ) , and body iron values tended to increase with age in boys , whereas the values remained stable in girls.conclusions:to ensure adequate iron stores , specific attention should be given to girls at european level to ensure that their dietary intake of iron is adequate .