all subjects were participants in the screening cohort of the dpt-1 study ( 5 ) who were screened for the presence of icas between february 1994 and october 2002 for potential study accrual to delay or prevent type 1 diabetes .
the current study group was comprised of first- and second - degree relatives of patients with type 1 diabetes ( 318 years of age ) who screened negative for all daas ( ica , ica512 , gad65 , and miaa ) at their first screening and returned for a rescreening .
miaa was measured on all subjects at their initial screening and used as inclusionary criteria to determine autoantibody - free status ; thereafter , miaa was only measured on a small proportion of the daa - negative subjects at rescreening .
autoantibody - negative subjects were rescreened at subsequent visits for ica , ica512 , and gad65 . for all other daas
, those aged 310 years were rescreened annually , and those aged > 10 years were screened biennially .
ica values were determined using the standard indirect immunofluorescence method using cryo - cut sections of frozen sections of human pancreas at the dpt-1 ica core laboratory ( gainesville , fl , and new orleans , la ) .
the ica assays had a specificity of 100% with a sensitivity of 74.4% ( 6 ) .
gad65 and ica512 were determined at the barbara davis center in denver , colorado , and measured simultaneously by combined gad65 and ica512 radioassay as previously described ( full - length gad65 and ica512bdc cdna clones ) ( 7 ) .
the assay was performed in 96-well filtration plates with autoantibody - bound [ h]gad65 and [ s]ica512 precipitated with protein a - sepharose .
the cut points were set at indices of 0.032 ( gad65 ) and 0.049 ( ica512a ) .
this assay had a specificity of 99% and a sensitivity of 83.7% in subjects 30 years of age with new - onset type 1 diabetes in the ids combinatorial autoantibody workshop ( 6 ) .
iaa values , using the microvolume requiring assay , were determined at the barbara davis center or the joslin diabetes center ( boston , ma ) ( 8) .
the sensitivity for the miaa assay was 74%/56% , and the specificity was 90%/98% for the denver and boston laboratories .
the correlation coefficient between both laboratories was r = 0.90 , p < 0.0001 , and the interassay cv was 16% .
the protocol was approved by the institutional review boards at participating locations , and all participants provided written informed consent .
data were analyzed using the sas software ( version 9.2 ; sas institute , cary , nc ) .
continuous variables were tested using t test for differences in means or wilcoxon rank - sum test for differences in medians .
cox proportional hazards models for an open cohort design were used to estimate 2-year risks for seroconversion and to determine significant predictors of seroconversion .
two - year risk estimates were chosen based on median follow - up time in this cohort .
efron s method for tied survival times were used in the cox analysis ( 9 ) .
selection of variables for the final model was accomplished by removing nonsignificant effects in a stepwise fashion .
the assumption of constant hazard function was assessed by examining the log cumulative survival plots .
ica values were determined using the standard indirect immunofluorescence method using cryo - cut sections of frozen sections of human pancreas at the dpt-1 ica core laboratory ( gainesville , fl , and new orleans , la ) .
the ica assays had a specificity of 100% with a sensitivity of 74.4% ( 6 ) .
gad65 and ica512 were determined at the barbara davis center in denver , colorado , and measured simultaneously by combined gad65 and ica512 radioassay as previously described ( full - length gad65 and ica512bdc cdna clones ) ( 7 ) .
the assay was performed in 96-well filtration plates with autoantibody - bound [ h]gad65 and [ s]ica512 precipitated with protein a - sepharose
. the cut points were set at indices of 0.032 ( gad65 ) and 0.049 ( ica512a ) .
this assay had a specificity of 99% and a sensitivity of 83.7% in subjects 30 years of age with new - onset type 1 diabetes in the ids combinatorial autoantibody workshop ( 6 ) .
iaa values , using the microvolume requiring assay , were determined at the barbara davis center or the joslin diabetes center ( boston , ma ) ( 8) .
the sensitivity for the miaa assay was 74%/56% , and the specificity was 90%/98% for the denver and boston laboratories .
the correlation coefficient between both laboratories was r = 0.90 , p < 0.0001 , and the interassay cv was 16% .
the protocol was approved by the institutional review boards at participating locations , and all participants provided written informed consent .
data were analyzed using the sas software ( version 9.2 ; sas institute , cary , nc ) .
continuous variables were tested using t test for differences in means or wilcoxon rank - sum test for differences in medians .
cox proportional hazards models for an open cohort design were used to estimate 2-year risks for seroconversion and to determine significant predictors of seroconversion .
two - year risk estimates were chosen based on median follow - up time in this cohort .
efron s method for tied survival times were used in the cox analysis ( 9 ) .
selection of variables for the final model was accomplished by removing nonsignificant effects in a stepwise fashion .
the assumption of constant hazard function was assessed by examining the log cumulative survival plots .
characteristics of the study population by daa development are presented in table 1 . of the 42,447 subjects
aged 318 years screened for daas in the dpt-1 study , 3,235 subjects ( 7.6% ) were autoantibody positive at the time of initial screening . of these ,
( table 2 , first column ) 1,454 subjects ( 3.4% ) were ica positive , 1,758 ( 4.1% ) were gad65 positive , 899 ( 2.1% ) were ica512 positive , and 1,094 ( 2.6% ) were miaa positive . at the initial screen , 39,212 subjects were autoantibody negative .
there was no significant difference in age ( p = 0.2964 ) or race / ethnicity ( p = 0.0798 ) between the two groups ( daa+ vs. daa ) .
there were small proportional differences between sexes ( p = 0.008 ) ( table 1 ) , and there was a significantly higher proportion of siblings of patients with type 1 diabetes ( p < 0.0001 ) in children positive for an autoantibody .
characteristics of the screened population aged 318 years data are median ( q1q3 ) and percent unless otherwise indicated .
number of subjects autoantibody positive and screened / rescreened by specific autoantibody * gad65 and ica512 were measured on 98% of those that returned for a rescreening .
rescreening not evaluated in this study . for those daa negative at the initial screen and who returned for at least one rescreening ( n = 11,813 ) , 469 ( 4.0% )
seroconverted to daa(s ) positive , 258 ( 2.2% ) to ica positive , 234 ( 2.0% ) to gad65 positive , and 99 ( 0.8% ) to ica512 positive ( table 2 ) .
these children were significantly younger ( p < 0.0001 ) in median age ( years [ quartile 1quartile 3 ] ) at the initial screen ( 7.1 years [ 5.110.0 ] vs. 8.2 years [ 5.511.6 ] ) compared with the remaining children who did not seroconvert ( n = 11,344 ) . median ( q1q3 ) time of follow - up for those who returned for a rescreening in the study was 27.1 months ( 20.149.1 ) .
there were no differences in race ( p = 0.39 ) , relation to proband ( p = 0.11 ) , or sex ( p = 0.42 ) among those who seroconverted and those that remained autoantibody negative throughout follow - up .
fifty - four percent ( n = 251 ) of the children who seroconverted did so by 11 years of age , with 75% seroconversion by 13 years of age .
there was no significant difference in the number of rescreens for those who seroconverted 10 versus > 10 years of age ( p = 0.76 ) .
median ages at seroconversion ( years [ q1q3 ] ) were similar with the exception that children seroconverted to gad65 at a significantly younger age than to ica ( p = 0.02 ) ( gad65 , 9.5 years [ 7.112.6 ] ; ica , 10.2 years [ 8.213.7 ] ; and ica512 , 9.9 years [ 7.313.5 ] ) .
for those individuals who developed multiple autoantibodies , gad65 seroconversion generally occurred earlier than ica or ica512 , with ica seroconversion usually occurring after ica512 .
if a child tested positive for two daas at the same testing , it was likely a combination of gad65 and ica512 , as the timing of each of these daas generally preceded the occurrence of ica , giving rise to two positive autoantibodies .
the 2-year risk for seroconversion by age showed significant decreases ( p < 0.001 ) with increasing age for all daas ( fig .
gad65 2-year risk had a significantly different slope of decline compared with ica512 ( p < 0.0001 ) and ica ( p <
0.0001 ) across all ages , with gad65 risk having a steeper slope for those aged 6 years and under .
ica risk was significantly higher than ica512 risk for all ages ( p = 0.003 ) .
the 2-year risk for any daa was significantly ( p < 0.0001 ) higher across all ages than for any two daas .
two - year risk of autoantibody seroconversion by autoantibody and development of any autoantibody(s ) by age ( years ) .
gad65 , gray dashed line ; ica , solid black line ; ica512 , black dashed line ; any autoantibody , black dotted line ; and any two autoantibodies , solid gray line . using cox proportional hazards models ,
the age at initial screening was identified as a significant predictor for risk of autoantibody seroconversion for all autoantibodies ( ica , p = 0.01 ; ica512 , p = 0.004 ; gad65 , p < 0.0001 ) . in univariate models
, siblings had a 46% ( hr 1.46 , 95% ci 1.062.03 ) greater risk of gad65 seroconversion ( p = 0.02 ) , and females had 21% ( 0.79 , 0.640.98 ) lower risk of ica seroconversion ( p = 0.03 ) .
race / ethnicity was not a significant predicator for autoantibody seroconversion in this cohort . in multivariable models adjusted for race , sex ,
relation to index proband , and rescreen interval , age at initial screening was the primary predicator of autoantibody seroconversion for any daa ( p < 0.0001 ) .
the risk of daa seroconversion greatly increased if the subject was already positive for another autoantibody .
daa seroconversion risk significantly decreased with increasing age for gad65 ( hr 0.90 , 95% ci 0.870.93 , p < 0.0001 ) , ica ( 0.96 , 0.930.99 , p = 0.01 ) , and ica512 ( 0.92 , 0.870.98 , p = 0.004 ) . for each 1-year increase in age in this cohort ,
the risk of any autoantibody seroconversion ( 0.95 , 0.920.97 , p < 0.0001 ) decreased by 5% and for two autoantibodies risk decreased by 13% ( 0.87 , 0.820.93 , p < 0.0001 ) .
lastly , 69 ( 15% ) of the 469 that seroconverted during rescreening developed multiple autoantibodies .
of the 69 , 45 ( 65% ) seroconverted to multiple autoantibodies at the same screening and 24 ( 35% ) developed multiple autoantibodies over time .
twenty - four of the 469 subjects ( 5% ) who daa seroconverted developed type 1 diabetes . at the time of diagnosis ,
8% were ica positive only , 4% were gad65 positive only , 4% were gad65 and ica512 positive , 17% were ica and gad65 positive , 21% were ica512 and ica positive , and 45% were positive for all three autoantibodies .
there were 19 subjects that developed type 1 diabetes who had no autoantibodies at the last screening before diagnosis ( median was 15 months prior to diagnosis ) .
the median age ( q1q3 ) at type 1 diabetes diagnosis was 12.4 years ( 9.714.7 ) .
autoantibody seroconversion rates are age - dependent among the first- and second - degree relatives of type 1 diabetic probands followed through childhood and adolescence in the dpt-1 , with a declining seroconversion rate with increasing age .
although , the risk of seroconversion for daas has been reported to be greatest in early life ( < 10 years ) ( 10 ) , risk of seroconversion exists throughout childhood and adolescence . while not in direct conflict with colman et al .
( 4 ) , the dpt-1 cohort provided additional information to possibly augment their recommendations to screen more than once before puberty .
gad65 and ica had the highest 2-year seroconversion risks , with gad65 carrying a greater risk for those in early childhood ( 6 years of age ) .
seventy percent of those daa positive in this cohort were identified at the initial screen , and rescreening efforts were successful at identifying an additional 10% of the expected 30% likely to seroconvert in the daa - negative cohort .
of all the daa - negative subjects eligible for rescreening ( n = 39,212 ) , the subjects that returned for rescreening ( n = 11,813 ) were similar to the subjects that did not return for rescreening ( n = 27,399 ) regarding age , sex , race , and relation to proband . by applying the absolute rate of seroconversion ( 469/11,813 ) to the entire negative cohort ,
the initial screen identified 3,235 ( 70% ) of the estimated 4,792 subjects likely to have seroconverted if the entire cohort was rescreened during the follow - up period .
the estimated cost for a daa screen ( including all autoantibodies ) is $ 70 .
therefore , the initial screen cost per seroconversion was an estimated $ 918 ( $ 70 for each of the 42,447 subjects at initial screen ) to identify 70% of those who were daa - positive versus $ 1,763 ( $ 70 for each of the 11,813 subjects rescreened ) for the remaining 10% ( n = 469 ) that were identified through the dpt-1 rescreening effort .
although economic realities may limit the ability to continually rescreen , strategies designed to capture those at highest risk are needed with the rapidly increasing incidence of type 1 diabetes worldwide and the highest observed annual rates in the youngest age - group ( 5.4% [ 4.86.1 ] for children age 04 years ) ( 11 ) .
the dpt-1 cohort data show that the majority of those that seroconverted were among the youngest and had the highest 2-year risk . as such ,
if we aim to identify the majority of subjects at risk for type 1 diabetes , it is necessary to continue screening for daas in early childhood , whereas screening in late adolescence and early adulthood will likely identify those with late - stage -cell decline in whom therapy is not as effective . based on these findings , annual rescreening for autoantibody seroconversion in relatives of type 1 diabetic probands is suggested for those in early childhood and adolescence and only once in late adolescence .
this strategy is more cost effective to optimize identification of subjects at risk for daas and eligible for participation in prevention trials . despite the important and novel observations described , this study s main limitation was that timing of rescreening was not uniform across subjects .
subjects 310 years of age were rescreened annually , whereas subjects > 10 years of age were rescreened biennially .
therefore , timing to seroconversion for those older than 10 years of age could occur anytime during a 2-year window .
this difference in the screening interval for those > 10 years of age could potentially underestimate the risk , where the time to daa seroconversion could be a year earlier than observed .
however , after assessment of the impact of this potential bias by adjusting for the screening cycle difference , age continued to be significantly associated with daa seroconversion independent of the screening cycle . also , it is likely the 2-year risks of daa seroconversion may be underestimated because of the median age at initial screening in this cohort ( 10 years ) ; however , this provided additional support to initiate screening strategies earlier in life .
additionally , iaa seroconversion was not evaluated in this study because of limited measures on only a very small proportion of those rescreened .
this limitation may be apparent in assessing risk for type 1 diabetes development , since iaas have been shown to be a significant indicator of risk , as have znt8 autoantibodies .
lastly , this study was not able to assess risk for type 1 diabetes because of limitations in type 1 diabetes development follow - up on the screened population in the dpt-1 .
although many studies have assessed the presence of autoimmunity in type 1 diabetic high - risk subjects , the data have been limited in assessing screening strategies for type 1 diabetes autoimmunity .
understanding the etiology of the preclinical period of diabetes by determining the time of autoantibody seroconversion may assist in identifying potential environmental triggers associated with the etiology of islet autoimmunity .
this study provides evidence that the rate of autoantibody seroconversion declines with age , varies throughout childhood and adolescence , and extends into early adulthood .
those in early childhood have the highest 2-year risk for any daa seroconversion ( 2.7% at 3 years ) , while the risk declines further from 1.8% at 10 years to 1.2% for those over 16 years of age .
these findings suggest starting screening in early childhood for those at increased risk ( i.e. , those with a relative with type 1 diabetes ) and screening annually for all daas ( including znt8 autoantibodies and iaas not measured in this study ) through early adolescence to capture the majority of those at risk for diabetes autoimmunity .
 objectivealthough type 1 diabetes autoimmunity frequently begins in childhood , little is known about the relationship between age and autoimmunity development . our aim was to determine the timing of seroconversion to diabetes - associated autoantibody ( daa ) positivity and risk in first- and second - degree relatives of patients with type 1 diabetes.research design and methodsstudy subjects were identified through the diabetes prevention trial - type 1 ( dpt-1 ) . 
 children 318 years of age ( n = 42,447 ) were screened for daas ; 1,454 were ica positive ( 10 jdf units ) , 1,758 were gad65 positive , and 899 were ica512 positive at the time of initial screening . 
 subjects who were initially antibody negative ( n = 39,212 ) were recalled for rescreening , and 11,813 returned for rescreening.resultsdaa seroconversion occurred in 469 ( 4% ) children ; 258 seroconverted to ica , 234 to gad65 , and 99 to ica512 . 
 the median time to seroconversion was 2 years . 
 the 2-year risk for daas was highest in early childhood . 
 for each 1-year increase in age in this cohort , the risk of any autoantibody seroconversion ( hr 0.95 , 95% ci 0.920.97 ) decreased by 5% , and for any two autoantibodies risk decreased by 13% ( 0.87 , 0.820.93).conclusionsrisk of autoantibody seroconversion among children followed in dpt-1 is age dependent . 
 younger children have the highest risk for daas , with the majority of children seroconverting by 13 years of age ( 75% ) . 
 this suggests that annual screenings should be started in early childhood and continued through early adolescence to identify the majority of subjects at risk for type 1 diabetes and eligible for prevention trials . 
RESEARCH DESIGN AND METHODS
Autoantibody assays
GAD65, ICA512, and mIAA
Statistical analysis
RESULTS
CONCLUSIONS