TRIGR - Trial to Reduce IDDM in the Genetically at Risk
Phase: NA
Conditions: Diabetes Mellitus, Type 1
Sponsor: University of Helsinki

 
 
 
PROTOCOL 
(Version April 2015) 
 
 
 
 
TRIGR 
Trial to Reduce IDDM in the Genetically at Risk 

Protocol – revised April 2015 
 
RESEARCH DESIGN AND METHODS 
 
This is a randomized double blinded intervention study using an intention to treat statistical analysis to 
compare the incidence of predictive autoantibodies and clinical diabetes in the two treatment groups. 
 
 
A. STUDY POPULATION 
 
Newborn infants who have a first degree relative (FDR) with type 1 diabetes (i.e. a mother, father or full 
sibling), and who meet the inclusion but not the exclusion criteria are recruited: 
 
Inclusion criteria: 
1. The biological parent and/or full (not half) sibling of the newborn infant has type 1 diabetes as 
defined by the World Health Organization 
2.   The infant’s parent or legal guardians give signed consent to participate 
 
Exclusion criteria: 
1. An older sibling of the newborn infant has been included in the TRIGR intervention  
2. Multiple gestation 
3. The parents are unwilling or unable to feed the infant cow’s milk (CM) based products for any 
reason (e.g., religious, cultural).  
4. The newborn infant has a recognizable severe illness such as those due to chromosomal abnormality, 
congenital malformation, respiratory failure needing assisted ventilation, enzyme deficiencies, etc. 
5. The gestational age of the newborn infant is less than 35 weeks  
6. Inability of the family to take part in the study (e.g. the family has no access to any of the Study 
Centers, the family has no telephone)  
7.  The infant has received any infant formula other than Nutramigen prior to randomization 
8. The infant is older than 7 days at randomization 
9.  No HLA sample drawn before the age of 8 days  
 
 
B. SAMPLE SIZE 
 
The sample size estimation presented in Table 1 is based on experiences from family studies analyzing 
the occurrence of autoantibodies in siblings of children with type 1 diabetes or in offspring of affected 
parents and progression to clinical disease in such young first-degree relatives. The data on the 
frequency of multiple (> 2) autoantibodies by the age of 6 years and the cumulative incidence of type 1 
diabetes by the age of 10 years are based on 82 young siblings from the DiMe Study carrying increased 
genetic risk for type 1 diabetes as defined according to the criteria in the Pilot-2 trial (106).  
 
Nineteen out of 325 offspring (5.9%) with increased genetic risk according to the criteria used in the 
Pilot-2 trial tested positive for at least one autoantibody by the age of 2.4 years in the German 
BABYDIAB study (107). Seventeen of these 19 subjects (89%) developed multiple autoantibodies 
during prospective observation.  Assuming a constant increase in autoantibody frequency from age 2 to 
 
1 

Protocol – revised April 2015 
age 6 years, the expected cumulative incidence of at least one antibody would be 14.8%  and that of at 
least two antibodies 13.0% by the age of 6, which is well within the 95% CI of the observed frequency 
of 20.7% of multiple autoantibodies in the young DiMe siblings.  
 
The cumulative incidence of at least one autoantibody by the age of 4 years was 10.6% in siblings and 
offspring of affected subjects in the DAISY study from Denver (108).  The frequency reached 37.9% in 
those carrying the DR3/4, DQB1*0302 combination.  Published data are not available allowing an 
estimation of the frequency of at least one or at least two autoantibodies in subjects with increased 
genetic risk as defined in the Pilot-2 trial.  Nevertheless a constant increase in the frequency of at least 
one autoantibody from age 4 to age 6 years, would result in an autoantibody prevalence of 15.9% by the 
age of 6 years in siblings and offspring irrespective of HLA-defined genetic risk. The DiMe study was 
used for the power calculation as it includes the largest long term follow-up of young relatives. 
 
The estimation that 2032 infants should be randomized for the trial (Table 1) is based on the following 
assumptions: 
1. a confidence level of 95%; 
2. a statistical power of 80%; 
3. a reduction of 40% in the hazard rate of type 1 diabetes in the intervention group; 
4. a drop-out rate of 20%; and 
5. a frequency of 10% of exclusive breast feeding (BF) up to the age of 6 months  
 
These figures represent a conservative estimate, since they are based on the lower 95% CI (7.6%) of the 
observed cumulative incidence of type 1 diabetes by the age of 10 years (15.4%) in young siblings with 
moderate (DQB1*0302/x) and slightly increased genetic risk (DQB1*02/y), and since the high risk 
(DQB1*02/0302) group was not considered in the calculations. In the Pilot-2 trial the prevalence of the 
high risk genotype (DQB1*02/0302) was about two times higher (absolute frequency 20%) among 
siblings of affected children than among offspring of affected parents. If also the young siblings carrying 
the high risk genotype in the DiMe Study should be included in the group at risk, the lower 95% CI 
would be 10.6%, which should result in a recommendation of 1920 infants to be randomized for the 
trial. However, fewer data from non-Finnish populations are available, explaining the caution of this 
sample size estimate. 
 
2 

Protocol – revised April 2015 
  
Table 1. Estimation of the sample size for the trial 
 
 Assumptions: 
• Confidence level:  
 
 
 
95% 
• Power: 
 
 
 
 
 
80% 
• Reduction in the annual frequency of multiple auto- 
     antibodies (> 2 AB) by the age of 6 years  
30, 40 or 50% 
• Reduction in the annual frequency of type 1 diabetes 
     by the age of 10 years 
 
 
 
30, 40 or 50% 
 
Frequency of > 2 AB 
SAMPLE SIZE 
by the age of 6 
30% effect 
40% effect 
 
50% effect 
  
I 
II 
III 
I 
II 
III 
I 
II 
III 
20.7% (*02 and/or *0302) 
1048 1132 1165 
558 
602 
626 
338 
364 
382 
18.5% (*0302/x, *02/y 
1196 1294 1330 
636 
688 
716 
386 
418 
438 
9.9% (lower 95% CI) 
2252 2438 2511 
1200 1300 1334 
732 
792 
832 
 
 
Frequency of type 1 
SAMPLE SIZE 
diabetes by the age of 10 
30% effect 
 
40% effect 
 
50% effect 
 
I 
II 
III 
I 
II 
III 
I  
II 
III 
18.3% (*02 and/or *0302) 
1330 1492 1536 
706 
794 
824 
428 
482 
502 
15.4% (*0302/x, *02/y) 
1548 1782 1834 
844 
948 
986 
512 
576 
606 
7.6% (lower 95% CI) 
3250 3658 3768 
1735 1952 2032 
1056 1190 1250 
 
Column I: Mathematical sample size 
Column II: Sample size corrected for a drop-out rate of 20% 
Column III: Sample size corrected in addition for a 10% frequency of exclusive BF up to the age of 6 
months. 
RECOMMENDATION: 2032 infants and families 
SCREENING TARGET: 4516 infants 
 
 
All statistical analyses will be based upon the total cohort of subjects randomized into the trial.  
Although data on some subjects may be missing at points in time, all relevant data available from each 
subject will be employed in the analyses.  In all principal analyses, subjects will be included in the group 
 
3 

Protocol – revised April 2015 
to which they were initially assigned and this assignment will not be altered based on a subject's 
adherence to the assigned treatment program.  Analyses of each outcome will include an assessment of 
possible differences among clinical centers.  Baseline variables that will be used in stratified analyses 
include gender, HLA risk category and other prognostic characteristics.  If differences in baseline 
characteristics are observed, analyses will be conducted of the effects of the treatments on outcomes 
adjusting for the potential confounding effects of these baseline characteristics.  If only a few such 
baseline characteristics are identified, analyses will be conducted stratifying for those characteristics.  If 
any more than a few baseline characteristics are identified, because of small sample size, it will be 
necessary that regression models be employed to adjust the treatment comparison for the confounding 
effects of those characteristics.  A number of subgroup analyses are planned to help identify individuals 
more likely to benefit from, or to be harmed by, the treatment.  In regard to treatment effects, definition 
of such subgroups will rely on baseline data, not data measured after randomization. Such subgroups 
might include:  gender, gender of the relative with type 1 diabetes, HLA risk group and other factors 
suspected to be associated with the event.  Exploratory data derived through subgroup analyses will 
serve primarily to generate new hypotheses for subsequent testing, and conclusions drawn from 
subgroup hypotheses not explicitly stated before data analysis will have less credibility than those from 
hypotheses stated in the protocol.  For safety monitoring, the Data Management Unit also will perform 
any and all analyses that are appropriate to identify subgroups that may be at significantly increased 
safety risk. The Kaplan-Meier method will be used to construct survival curves and the logrank statistic 
used to compare treatment arms with respect to time until the development of autoimmunity or type 1 
diabetes.   
 
We therefore aim to randomize 2032 infants with increased genetic risk as defined in the genetic 
screening section (E).  To achieve that number 4516 infants must be screened assuming a frequency of  
45% of the genotypes conferring increased risk. The observed prevalence of risk genotypes was 50% 
among the 471 infants screened for the Pilot-2 trial in Finland. An enrollment scheme for the trial proper 
is presented in Figure 1. This flow sheet indicates that the trial requires initial access to 5806 
pregnancies. This should provide at least 4936 families with consent to participate in the trial before the 
birth of the child provided that the consent rate is 85%. With an exclusion rate of 8.5% after birth based 
on the exclusion criteria approximately 4516 infants will be available for genetic screening. The 
observed exclusion rate in the Pilot-2 trial was 8.6%. 
 
Infants will be recruited over a four-year period and the planned follow-up will be 6 years after the last 
infant has been accrued for the antibody endpoint and 10 years after the last infant is accrued for the 
type 1 diabetes end point. Thus, all subjects will have at least 10 years of follow-up. 
 
 
4 

Protocol – revised April 2015 
Figure 1.  Enrollment scheme for TRIGR 
Newborn infants
N = 5806
Consent to screening
(approximately 85%)
N ≈4936
Excluded
(approximately 8.5%)
n ≈420
Newborn infants, who fulfill the inclusion criteriaand whose parents have given their consent, N ≈4516
Risk genotypes :
HLA-DQB1*02/0302
Other genotypes
HLA-DQB1*0302/x (x ≠DQB1*02, *0301, *0602)
n ≈2484
HLA-DQA1*05 -DQB1*02/y (y ≠DQA1*0201 -DQB1*02, DQB1*0302, *0301, *0602, *0603)            EXCLUDED
HLA-DQA1*03 -DQB1*02/y (y ≠DQA1*0201 -DQB1*02, DQB1*0302, *0301, *0602, *0603)
n ≈2032
Experimental group
Control group
n≈1016
n≈1016
Expected to complete the trial
Expected to complete the trial
n ≈813
n ≈813
Randomization
Withdrawn 20%
n ≈406
GENOTYPING
 
 
 C. RECRUITMENT 
 
Recruitment is carried out in six major centers in USA, in 18 centers in Canada, in 12 European 
countries, and three centers in N.S.W., Australia.  A list of participating countries and recruitment 
estimates are presented in Appendix 1. The figures represent an estimate of eligible pregnant women 
available in each country. The background of these estimates is that the appraisal of the acceptance rate 
(best scenario 70-80%, average 60%) may vary from country to country, and accordingly the total 
recruitment figures are a rough estimate. According to the figures given in Appendix 1 about 65% of the 
participants will be recruited in North America and 35% in Europe + Australia.  The recruitment will be 
continued past the initial 4-year period, if we have not obtained 2,032 newborn infants with increased 
genetic risk to the series by that time.  
 
 
To facilitate recruitment and to minimize any possibility of unintentional exposure to CM protein, every 
attempt is made to identify eligible families before the child is born.  Written consent is obtained at this 
time; the child will participate after birth if he/she meets the inclusion but not the exclusion criteria.  
Families not identified until just prior to the onset of maternal labor are approached after birth and 
written consent is obtained at that time. 
 
5 

Protocol – revised April 2015 
 
 
Mothers with type 1 diabetes are identified during pregnancy via hospitals monitoring pregnant women 
with type 1 diabetes.  Fathers with type 1 diabetes are identified by  (i) available history or data already 
in the medical records of pregnant women, (ii) interviewing women at prenatal maternity clinic visits, 
and (iii) existing registries of type 1 diabetes in some centers.  Pregnant women already having one or 
more children affected with diabetes are approached through various diabetes clinics.  Children with 
type 1 diabetes are usually followed by pediatricians or pediatric endocrinologists, who are aware of a 
pregnant mother. The identification of such mothers is arranged through these physicians.   
 
 
Experience gained in the Pilot-2 study of this project is utilized to optimize the efficacy of the 
recruitment in the study proper (use of diabetes societies, information in mass media etc.).  In that pilot 
study we also found that weekly staff meetings and continuing recruitment during weekends and 
vacation periods substantially raises recruitment efficiency. Leaflets, distributed to “candidate” mothers, 
describing the project can be very helpful. 
 
 
D. SUBJECT ALLOCATION 
 
Randomization takes place before birth or immediately after the birth of the child. The research assistant 
or investigator obtains the formula allocation code from the Data Management Unit (DMU) by 
completing the Randomization Form electronically.  Subjects who meet the inclusion and do not meet 
exclusion criteria are randomized as follows: randomization in each strata will be within four blocks. 
Subjects will receive either the test formula, casein hydrolysate (Nutramigen™, Mead Johnson 
Nutritionals), not containing antigenic CM protein, or a CM protein containing control formula which 
has an addition (20 %) of Nutramigen, whenever breast milk is not available.  Any subject requiring 
supplemental feeding prior to randomization (e.g. infants born at night or on weekends)  is given banked 
breast milk or Nutramigen.  The randomization code will be kept by the manufacturer and it will be 
opened when TRIGR study is completed. 
 
 
Cord blood is obtained from newborn infants whenever possible or alternatively a heel prick is 
performed to obtain capillary blood as soon as possible after birth, at the latest at the age of 7 days.  The 
blood sample is forwarded to the continental genotyping center and screening performed for the 
presence of the genotypes listed below indicating an increased genetic risk. Results are entered into the 
Central Data Base immediately when available (within 2 weeks after the sampling) and are sent by e-
mail from DMU to the national coordinating centers and/or to the local study center in question and to 
the Study Monitors.  Only subjects with these genotypes are included in the nutritional prevention trial; 
all other subjects are withdrawn from the study at that time; their parents are told that genetic screening 
suggests that their child does not fall into the increased genetic risk group for type 1 diabetes and they 
are appropriately thanked for their participation.  It is emphasized that their child might still develop 
diabetes despite not meeting the eligibility criteria. 
 
 
 
6 

Protocol – revised April 2015 
 
 
E. GENETIC SCREENING 
 
The options for genetic screening vary between the extremes of a very high risk population requiring a 
small sample size (e.g. DQA1*0301-DQB1*0302/DQA1*0501-DQB1*02 heterozygotes), and a lower 
average  risk population with larger sample size (e.g. newborn infants with a FDR but with no additional 
genetic selection applied).  Based on anticipated rates of identification of eligible newborn infants and a 
feasible enrollment period, we propose to base the selection on the presence of the following genotypes, 
representing different risk categories: 
 
1.  HLA-DQB1*0302/DQB1*02 
2.  HLA-DQB1*0302/x (x not DQB1*02, DQB1*0301 or DQB1*0602) 
3.  HLA-DQA1*05-DQB1*02/y (y not DQA1*0201-DQB1*02, DQB1*0301, DQB1*0302, 
DQB1*0602 or DQB1*0603) 
4.  HLA-DQA1*03-DQB1*02/y (y not DQA1*0201-DQB1*02, DQB1*0301, DQB1*0302, 
DQB1*0602 or DQB1*0603)  
 
The HLA-DQB1 genotyping used in pilot studies has been completed by additional DQA1 typing in 
samples positive for DQB1*02 allele but without protective DQB1 alleles.  These samples are analyzed 
for the presence of DQA1 alleles DQA1*0201, DQA1*03 and DQA1*05.  This procedure allows the 
distinction between the diabetes risk associated (DQA1*05-DQB1*02 and DQA1*03-DQB1*02) and 
non-associated haplotypes (DQA1*0201-DQB1*02).  The effect of this further step is relatively small in 
the populations of Northern European descent, but more important in some Mediterranean and Black 
populations.  
 
The efficiency of this new screening procedure was tested in an enlarged series of 289 familial cases of 
type 1 diabetes in Finland as well as in the newborn infants recruited for the Pilot-2 study.  85.8% of 
familial cases were positive for the selected genotypes which on the other hand were present in 50% of 
the 471 newborn infants with a FDR affected by type 1 diabetes.  If an approximate figure of 10% is 
used for the risk of a FDR to develop multiple autoantibodies before the age of 6 yrs, positive predictive 
values delineate a risk of 17.1% for those with risk genotypes compared to a risk of 2.8% of those 
without a risk genotype.  This figure is close to the 20.7% proportion given in Table 1 for the positivity 
of at least two autoantibodies by the age of 6 years.   
 
The selected risk genotypes are present in 81.0% of Finnish children with type 1 diabetes and in 22.5% 
of the background population, the higher frequency in familial cases confirms our earlier observations 
(4).  We do not have available data on familial cases and newborn infants with a FDR with type 1 
diabetes in other populations, but comparative analyses of genotype frequencies in patients with type 1 
diabetes and healthy controls indicate that the genotyping protocol would function with quite similar 
efficiency in various populations to enrich high risk subjects for the study (Table 2).  Several published 
data also support this view, although exact proportions cannot be deduced because of the lack of detailed 
genotype frequencies (5-7).   
 
 
 
 
 
7 

Protocol – revised April 2015 
Table 2.  The HLA risk genotypes in patients and healthy controls from various populations 
 
 
Population 
Type 1 
Diabetes 
%              N 
              studied 
Healthy  
Controls 
%               N 
               studied 
 
Reference 
Estonia 
78.4 
97 
14.1 
269 
Nejentsev et al.:Tissue Antigens 1998:52:473 
Finland 
81.0 
316 
22.5 
1000 
Ilonen et al.: Eur. J. Immunogenet. 2000 27:225 
Hungary 
87.5 
112 
21.0 
210 
Hermann et al., personal communication 
Italy (Sardinia) 
90.0 
100 
40.0 
100 
Songini & Ilonen, personal communication 
The Netherlands 
77.6 
205 
26.9 
840 
Schipper et al., personal communication 
USA (Puerto Rico) 
78.9 
114 
26.9 
108 
Santiago, Trucco & Frazer, personal 
communication  
 
 
 
F. STUDY FORMULA COMPOSITION, LABELLING AND DISTRIBUTION 
 
Each study formula is either a nutritionally complete infant formula, "Nutramigen" powder, 
manufactured by one company (Mead-Johnson Nutritionals, Evansville, IN, USA) containing 
extensively hydrolyzed casein as the protein source, vegetable oils as the fat source, and glucose 
polymers and modified starch as the carbohydrate source, or a control formula which is a mixture of 
commercial routine CM-based formula powder made by the same company plus casein hydrolysate 
powder in a 4:1 ratio designed to mask the flavor and smell distinctions between the two study formulas. 
The casein hydrolysate has been shown to reduce diabetes frequency in the NOD mouse (8) and BB (9) 
rat models.  The Study Formula contains all the nutrients infants need. Their nutritive value is analogous 
to the regular infant formulas used in the TRIGR countries.   
The formula has four different code numbers and has been packed in four different colours - two colors 
for test formula and two colors for control formula.  The 4-color coding scheme has been extensively 
tested in formula studies, it aids the blinding process, provides a hard control for randomization during 
data analysis, and avoids accidental mis-shipments as the families recognize “their” color.  Only Mead 
Johnson Nutritionals knows which colors correspond to test and control formulas.  Subjects are allocated 
to receive one color formula, which is maintained throughout the study.  Each center maintains a reserve 
supply of each "color" formula and is responsible for ensuring that the home supplies of each participant 
are maintained.  The company provides the coded formulas free of charge, whereas the shipping costs 
are paid by the study grant.  
 
 
G. IMPLEMENTATION OF INTERVENTION 
 
All recruited mothers are encouraged to breast-feed; the newborn infants are randomized before birth or 
as soon as possible after birth so that any elective formula supplementation or weaning by the mother 
will be done with the appropriate study formula.  The duration of the intervention will be until at least 6 
months of age.  If the mother chooses to exclusively breastfeed up to the age of 6 months she is advised, 
 
8 

Protocol – revised April 2015 
thereafter, if milk supplementation is needed, to give the study formula until the age of 8 months.  
Similarly, if exclusive BF lasts for 5 months, the infant would receive study formula for 2 months until 
the age of 7 months.  Infant feeding practices are altered as little as possible by the trial.  In particular, 
BF practice(s) is entirely at the discretion of participating mothers.  
 
The rationale for the 6-8 month intervention period is based on the following considerations: in early 
infancy the child receives the major part of energy in liquid form, either as human milk or CM-based 
formula.  The period chosen provides a reasonable and practical safety margin over the 2-3 months 
"vulnerable" time when the gut is permeable to proteins, as suggested in several epidemiological studies 
(reviewed in ref. 10). 
 
Dietary information 
Dietary advice is given by a member of the study team at the first contact with the family after 
randomization, at the 2 week, 1, 2, 4 and 5 month telephone calls from the center to the mother, and at 7 
and 8 months for those infants who continue in the intervention beyond the age of 6 months, and at the 3 
and 6 month visits.  The families receive both written and oral instructions about infant feeding during 
the intervention period.  Parents will be given a pamphlet which describes the sequence and amounts of 
food recommended at specific ages, according to local guidelines.  It is important to avoid any conta-
mination of the dietary intervention with sources of CM protein (including milk products, beef, and 
veal) contained in foods ingested by the infant.  Thus, parents are provided with a list of all solid foods 
giving choices of brand names which can be given to the infant and which do not contain CM protein; 
they are also provided with a list of foods to be avoided, as they contain CM protein.  Dietary advice 
leaflets, dietary forms and questionnaires are translated into relevant languages and adapted to national 
practices.  The TRIGR Manual of Operations contains detailed instructions about the dietary interviews 
and forms.  Post-intervention diets follow generally accepted practices.  Designated personnel will be 
available during working hours to deal with urgent nutritional questions by telephone (e.g. feeding 
intolerance). 
 
Monitoring of compliance and retention strategy 
The diet of the infant and the compliance with the avoidance of CM proteins are assessed at the delivery 
hospital by interview, by a telephone interview when the infant 2 week-old, 1, 2, 4, and 5 month-old, 
and by an interview during the visits at the age of 3, 6 and 9 months.  Those families, whose infants 
continue in the intervention beyond the age of 6 months, are in addition interviewed by phone, when the 
infant is 7 and 8-month-old.  The dietary interviews are similar at all stages. We ask the families about 
the age at the end of exclusive and total breastfeeding. We have several questions on the use of Study 
Formula:  Age when the feeding was started and age when regular, daily use started, whether the infant 
receives Study Formula at the time of the interview, how much Study Formula the infant is receiving per 
feeding, how much Study Formula the family has available at home at the time of the interview, and 
whether they need more or not. In the interview form we also have questions on the frequency of use of 
breast milk, Study Formula and other foods.  The frequency of use of foods to be avoided during the 
intervention period is also asked. The Study Centers record the amount of Study Formula given to the 
family, and all the unused formula must be returned to the local study center the clinical visit closest to 
the end of the intervention period  (at the 6 or 9 month visit).  Compliance with the avoidance of CM 
proteins is also assessed by measuring antibody levels to CM proteins from sera at 3 and 6 months, and 
the results are analyzed by the Data Safety and Monitoring Board.  We do not intend to employ serum 
titers of antibodies to CM proteins as a surrogate of islet cell damage.  
 
9 

Protocol – revised April 2015 
 
The Nutrition Epidemiology Unit (Dr. Suvi M. Virtanen, National Institute for Health and Welfare, 
Helsinki, Finland) of the International Coordinating Center is responsible for the education and 
supervision concerning the assessment and maintenance of the dietary compliance and the dietary advice 
given by the centers involved.  A continuous quality control system is implemented in each center for 
the data collection and content, especially to ensure that the delivery of dietary advice and the 
measurement of dietary compliance function properly.  A specially trained Study Monitor supervises the 
implementation of these measures.  A Nutrition Fellow at the Nutrition Epidemiology Unit oversees the 
dietary intervention closely supervised by Dr. Virtanen, by means of data collected, reports from the 
monitors, and continuous contacts with the Study Centers. 
 
 
H. STUDY ASSESSMENTS 
 
Baseline  
After the delivery medical and perinatal history of the infant and mother (including birth weight and 
gestational age) and the results of the newborn physical examination are recorded on the case report 
forms.  
 
 
Follow-up Period during and after Intervention 
The subjects visit the research center or have a home visit at the age of 3, 6, 9, 12, 18 and 24 months, 
and at 3, 4, 5, 6, 7, 8, 9 and 10 years of age, or when clinical diabetes develops.  
 
Protocol change in 2011: The follow-up of the subjects will be continued until the youngest participant 
reaches the age of 10 years. The oldest participants will then be 14-year old. During the prolonged 
follow-up period the follow-visits will take place annually at the ages of 11, 12, 13 and 14 years.  The 
presence or absence of diabetes is determined according to the criteria outlined below (see Outcome 
Assessment, below).  Clinical findings at each visit (e.g. weight, height) are recorded on the case report 
forms.   
 
Protocol Change in 2015:  Puberty will be assessed based on hormonal analyses. Serum estradiol will be 
analyzed in girls and serum testosterone in boys starting from the age of 8 years. In addition girls will be 
asked about their age at menarche and the use of medication containing estrogens and/or androgens 
starting from the age of 10 years.  
 
Blood for serology (diabetes-associated autoantibodies and CM antibodies) is drawn after the 
application of an analgesic ointment on the venipuncture site at the above mentioned visits and serum 
samples are stored centrally.  In addition, the 3 and 6 month serum samples are available for use in the 
assessment of dietary compliance.  All serum samples are aliquoted, and the samples are stored at -70 C° 
in the TRIGR Core Laboratory in Helsinki. Local measurement of plasma glucose and glycated 
hemoglobin takes place at 12, 18, 24 months, and 3, 4, 5, 6, 7, 8, 9 and 10 years. During the prolonged 
follow-up period these measurements will be performed at the age of 11, 12, 13 and 14 years.  The 
specimens for plasma glucose are preferentially taken 1-2 hours postprandially.  If the glycated 
hemoglobin is higher than the reference range, an OGTT may be required to confirm the diagnosis as 
below.  An OGTT will be performed at the age of 6 and 10 years in all study subjects. In addition, an 
 
10 

Protocol – revised April 2015 
OGTT will be performed in all study subjects at the last follow-up visit in 2016-2017.   A heparin blood 
sample (3-5 ml blood depending on age) is obtained at each sampling time for the isolation of 
mononuclear cells. These cells are sent fresh to central labs (Professors Outi Vaarala, Helsinki, Finland 
and Hans-Michael Dosch, Toronto, ON, Canada) and used for mechanistic studies. An EDTA blood 
sample, 4 ml, for DNA extraction will be collected from all study participants. Samples will be obtained 
in association of collection of blood samples for autoantibody analyses once at the age of 5 years or 
later. In the case of children who already have developed type 1 diabetes or have dropped-out from the 
follow-up a separate sample will be collected if possible.  
 
Protocol Change in 2015:  After TRIGR, any remaining samples will be placed into the National 
Institutes of Health (NIH) repository for future studies related to type 1 diabetes and its complications.  
They will be stored there indefinitely without names or any other identifying information on them.  
Once the samples are in the repository, subjects will not be able to have them removed.  Researchers 
must first get permission from the NIH to use samples from the repository. 
 
A summary and timetable of the various events are given in Table 3. 
 
 
11 

Protocol – revised April 2015 
 
Table 3. Study schedule 
 
  
0 mo* 
½, 1, 2 mo 
3 mo 
4, 5 mo 
6 mo 
7 mo 
8 mo 
9 mo 
12 mo 18 mo 
2 yr 
3 yr 
4 yr 
5 yr 
6 yr 
7 yr 
8 yr 
9 yr 
10 yr 
11 - 14yr 
Call 
Visit 
Call 
Visit 
Call 
Call 
Visit 
Visit 
Visit 
Visit 
Visit 
Visit 
Visit 
Visit 
Visit 
Visit 
Visit 
Visit 
Visit 
Contact 
number 
1 
2-4 
5 
6-7 
8 
8B 
8C 
9 
10 
11 
12 
13 
14 
15 
16 
17 
18 
19 
20 
21-24 
Weight and 
height 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Blood 
Specimens 
   - Cord  
     blood 
X 
   - Venous  
     blood 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
   - Heparin  
     blood 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
   - Blood  
     glucose 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
   - HbA1c 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
   - EDTA  
     blood 
 
 
 
 
 
 
 
 
 
 
 
 
 
X*** X*** X*** X*** X*** X*** 
 
Dietary 
Interview 
X 
X 
X 
X 
X 
X 
X 
X 
Dietary 
counselling 
X 
X 
X 
X 
X 
X 
X 
Delivery of 
Study 
Formula 
X 
+ When  
   
needed 
Examination 
by Study 
Doctor 
X 
X 
X 
X 
X 
X 
X 
X** 
OGTT 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
X 
  
  
  
X 
X** 
 
 
* Contact number 1 takes place in the delivery hospital but in case of home deliveries it is arranged at home 
** OGTT and examination by the Study Doctor will be performed in all study subjects at the last follow-up visit in 2016-2017.  
*** An EDTA blood sample, 4 ml, for DNA extraction will be collected from all study participants once at the age of 5 years or later 
 
12 

Protocol – revised April 2015 
 
Outcome Assessment  
The major outcome for the first phase will be the frequency of type 1 diabetes-associated autoantibodies 
and/or the development of clinical diabetes by the age of 6 years.  The outcome of the second phase will 
be the manifestation of diabetes by the age of 10 years.  The manifest diabetes outcome is assessed as 
the proportion of subjects in each group who develop type 1 diabetes, as well as age at diagnosis.  These 
subjects will be classified as having type 1 diabetes if they fulfill one of the following criteria: 
 
1)  Symptoms + a single random plasma glucose ≥ 11.1 mmol/l = 200 mg/dl (venous blood glucose > 
10.0 mmol /l = 180 mg/dl)  
2)  If the child has no symptoms the diagnosis requires  
a)  A raised random plasma glucose reading ≥ 11.1 mmol/l = 200 mg/dl (venous blood glucose ≥ 10.0 
mmol/l = 180 mg/dl) on two occasions or  
b)  A raised fasting plasma glucose readings ≥ 7.0 mmol/l = 126 mg/dl (venous blood glucose ≥ 6.1 
mmol/l = 110 mg/dl) on two occasions or  
c)  A diabetic oral glucose tolerance test by WHO criteria  
• fasting venous plasma glucose ≥ 7.0 mmol/l = 126 mg/dl (fasting venous blood glucose ≥ 6.1 
mmol/l =110 mg/dl) on two occasions 
 
 
or  
• 2 hour venous plasma glucose ≥ 11.1 mmol/l = 200 mg/dl (2 hour venous blood glucose ≥ 10.0 
mmol/l = 180 mg/dl)] on two occasions.   
 
Accordingly a second OGTT should be performed, if the first one is diabetic.  There should be 
an interval of at least one week between these two OGTTs. 
 
The diabetes-associated autoantibody data will be disclosed when the autoantibodies have been assayed 
after the 6-year visit, and the family will be informed by the Study Center as soon as the autoantibody 
results have become available. 
 
I. METHODOLOGY TO BE APPLIED 
 
Methodology for Genetic Screening 
The procedures used for HLA-DQ typing were specifically developed for screening relevant DQB1 and 
DQA1 alleles (11, 12).  EDTA treated cord blood is collected. Alternatively capillary blood is taken 
postnatally if cord blood is not available. The blood samples are sent by air mail or special delivery from 
the U.S. and Canadian centers to the tissue typing laboratory of Professor M. Trucco, University of 
Pittsburgh, PA, USA, and from the various European and other centers to the tissue typing laboratory of 
Dr. J. Ilonen, University of Turku, Finland.  According to experiences from the Pilot-2 study of the 
project (Finland, Sweden, Estonia and Hungary), the result of the genetic screening is available within 2 
weeks to assess the eligibility of the infants.  The results are entered into the Central Data Base 
immediately when available, and the Study Center is alerted by e-mail.  
 
The polymorphic gene region is amplified in a microtiter format using biotinylated primers. The 
amplification product is subsequently transferred to streptavidin coated microtiter plates where it is 
bound by solid phase streptavidin. After denaturation the hybridization reaction is performed with a 
mixture of lanthanide (Europium, Samarium or Terbium) labeled sequence specific oligonucleotide 
 
13 

Protocol – revised April 2015 
 
probes and specific hybridization signals detected by time-resolved fluorometry after repeated washes 
and the addition of enhancement solution. 
 
Methodology for Autoantibodies 
We will use islet cell antibodies (ICA), insulin autoantibodies (IAA), antibodies to the 65 kD isoform of 
glutamic acid decarboxylase (GADA) and antibodies to the protein tyrosine phosphatase related IA-2 
molecule (IA-2A) as humoral markers of β-cell autoimmunity.  All autoantibody analyses will be 
performed in the laboratory of Professor M. Knip, University of Helsinki.  His laboratory has a long 
experience and acknowledged expertise in the analysis of diabetes-associated autoantibodies.  The 
laboratory will pay particular attention to quality control procedures of the assays for autoantibodies.  
All samples are bar-coded, and the analyses will be performed on a blinded basis.  Samples from the 
same individual will, however, be stored in such a way that they can be run in the same assay.    Samples 
will be analyzed in the following assay rounds: the first including samples from the baseline visit (0 
month visit) up to the 2-year visit, the second from the 3-year visit to the 6-year visit, and after the 6-
year visit annually visit by visit. Possible assay drift over time will be monitored by analyzing blindly 
with each assay three standards (low, medium and high antibody levels) once a month.  An assay drift 
exceeding 10% is considered unacceptable. The laboratory has completed a process of collecting 
optimal standards with sufficient volume for a period of 15 years. 
 
ICA are analyzed with a standard immunofluorescence assay performed on sections of frozen human 
pancreas from a blood group O donor (13).  Fluorescein-conjugated anti-human IgG (Sigma, St Louis, 
MO, USA) is used to detect ICA.  All initially ICA-positive samples are re-tested to confirm antibody 
positivity.  End-point dilution titers are identified and the results are expressed in Juvenile Diabetes 
Foundation (JDF) units relative to an international reference standard (14).  The detection and cut-off 
limit is 2.5 JDF units.  The sensitivity of this ICA assay was 100%, the specificity 98% the validity 98%, 
and the consistency 98% in the most relevant international standardization round. 
 
IAA are measured with a micro-assay, modified from Williams et al. (15).  According to the assay 
protocol endogenous insulin is not removed before the assay.  Immune complexes are precipitated with 
Protein A Sepharose (Pharmacia Biotech, Uppsala, Sweden) after incubation for 72 h of the serum 
sample (5 µl/well) with mono 125I-(TyrA14)-labeled human insulin (Amersham, Little Chalfont, Bucks, 
UK) in the presence or absence of an excess of unlabeled insulin.  The volume of the incubation reaction 
is doubled by adding the reaction buffer (TBT; 50 mM Tris, pH 8.0, 1 % (v/v) Tween 20).  After 
thorough washing with the reaction buffer the samples are transferred from the deep well plates to 
microtiter plates, scintillation liquid is added and the bound activity measured with a liquid scintillation 
counter (1450 MicroBeta Trilux; Perkin Elmer Life Sciences Wallac, Turku, Finland).  The specific 
binding is expressed in relative units (RU) based on a standard curve run on each plate using the 
MultiCalc™ software program (Perkin Elmer Life Sciences Wallac).  The standard curve is constructed 
from nine serial dilutions of a serum from a patient with a high IAA titer and a serum of an IAA-
negative subject.  The negative human serum is considered as the lowest point of the standard curve.  
The cut-off limit for IAA positivity is set at the 99th centile in 371 non-diabetic Finnish subjects.  
Samples with an initial IAA level exceeding the 95th centile are reanalyzed to verify the antibody status.  
The disease sensitivity of this assay was 35 % and the disease specificity 100 % based on 140 samples 
included in the Multiple Autoantibody Workshop (16). 
 
 
14 

Protocol – revised April 2015 
 
GADA are detected in an immunoprecipitation radioligand assay (17, 18).  The recombinant plasmid 
pGEM3 encoding the whole 65 kD form of the GAD protein (585 amino acids) is propagated in E. coli 
JM 109 by standard techniques.  The GAD65 protein is produced by in vitro transcription and 
translation of the purified plasmid using the TNT Coupled Reticulocyte Lysate System (Promega, 
Madison, WI, USA) in the presence of 35S-methionine (Amersham). Unincorporated 35S-methionine is 
removed by gel chromatography on NAP-5 columns (Pharmacia Biotech).  Sera (2 µl) are incubated 
overnight at + 4 °C with approximately 20,000 cpm human GAD65 in a total volume of 50 µl TBST.  
To isolate the immune complexes, 10 µl Protein A-Sepharose® CL-4B (Pharmacia Biotech) is added the 
following day.  A scintillation counter is used to count the amount of precipitated immune complexes.  
The results are expressed in relative units (RU) based on a standard curve run on each plate using a 
commercial software program (MultiCalc, Perkin Elmer Life Sciences Wallac).  The cut-off limit for 
antibody positivity is set at the 99th percentile in 373 non-diabetic children and adolescents.  All samples 
with an initial GADA level between the 97.5th and 99.5th percentiles are reanalyzed to verify the 
antibody status.  This assay had a disease sensitivity of 69% and a specificity of 100% based on 140 
samples included in the 1995 Multiple Autoantibody Workshop (16).  
 
IA-2A are detected in a similar radiobinding assay (19).  The recombinant plasmid pSP64poly(A) 
encoding the intracellular portion of the full length IA-2 protein, including amino acids 605-979 is 
propagated in E. coli JM 109 cells.  The radioactive IA-2 protein is produced with the TNT Coupled 
Reticulocyte Lysate System (Promega) by in vitro transcription and translation of the purified plasmid in 
the presence of 35S-methionine.  Sera are incubated overnight at +4 °C with 10,000 cpm of labeled IA-2 
protein.  Protein-A Sepharose® (Pharmacia Biotech) is used to isolate the immune complexes on the 
following day.  After thorough washing the radioactivity of the samples is measured by a liquid 
scintillation counter (1450 Microbeta® Trilux, Perkin Elmer Life Sciences Wallac).  The results are 
expressed in RU based on a standard curve constructed from the dilution of a pool of strongly positive 
samples and a pool of negative samples.  The standard curve is run on each plate.  A subject is 
considered IA-2A positive, if the serum antibody levels are equal to or exceed the 99th percentile in 374 
non-diabetic Finnish children and adolescents.  Samples with an initial IA-2A level between the 97.5th 
and 99.5th percentiles are reanalyzed to verify the antibody status.  The disease sensitivity of this assay 
was 62% and the specificity 97% based on 140 samples included in the 1995 Multiple Autoantibody 
Workshop (16). 
 
Methodology for Antibodies to Cow's Milk Proteins  
CM antibodies (IgG, IgA and IgM), β-lactoglobulin antibodies (IgG and IgA), α-casein antibodies (IgG 
and IgA) and BSA antibodies (IgG and IgA) will be measured with a modification of the original ELISA 
technique by Dr. E. Savilahti, Helsinki, Finland. 
 
Microtitre plates (MaxiSorp®, Nunc A/S, Roskilde, Denmark) are coated with one of the following 
antigens: 1. adapted liquid CM formula (Tutteli®, Valio Ltd, Helsinki, Finland) defatted,  diluted (1:500 
in carbonate buffer, pH 9.6) bovernight,  2. bovine β-lactoglobulin (Sigma Pharmaceuticals, St. Louis, 
MO, USA), at a concentration of 1 mg/ml in carbonate buffer, pH 9.6, overnight, 3. BSA (2 mg/ml, 
grade V, Sigma), 4. α-casein (Sigma) 2 mg/ml in phosphate buffered saline (PBS), pH 7.4.  Thereafter 
they are incubated overnight.  Wells are blocked either with 0.5% sheep serum (for anti-CM and β-
lactoglobulin assays) or with 1% gelatin in PBS pH 7.4 (for BSA and α-casein assays).  Serum samples 
are diluted in the blocking buffers.  Triplicate dilutions for assays are 1:20 for β-lactoglobulin, 1:40 for 
CM and BSA and 1:100 for α-casein.  In assays for CM and β-lactoglobulin antibodies, plates are 
 
15 

Protocol – revised April 2015 
 
incubated with serum dilutions overnight at room temperature, in assays for BSA and α-casein at 37 °C 
for 1 hour.  After washing, 100 µl alkaline-phosphatase-conjugated affinity purified rabbit F(ab’)2 anti-
human IgG, IgA or IgM antisera (dilutions between 1:600 to 1:1200) (Dako A/S, Glostrup, Denmark) 
are added  for 60 minutes at 37 °C.  After washing, 100 µl of p-nitrophenyl-phosphate substrate, 2 
mg/ml in diethanolamine buffer, pH 10.0, (Medix Biochemica, Helsinki, Finland) is added.  The 
reaction is stopped after 30 minutes with 100 µl 1 M NaOH.  The end point measure of OD405 nm is 
obtained in a semi-automatic multiwell photometer (Titertek Multiscan®, Elflab Inc., Helsinki, Finland).  
The mean value of two absorbances for wells coated with blocking solution is subtracted from the mean 
value for the three absorbances in antigen-coated wells.  Results are subjected to point-to-point analysis 
in a computerized photometer using 2-fold serial dilutions of a high titer standard serum as reference.  
Sample dilutions must fall within the linear part of the standard curve, and antibody levels are expressed 
as percentages of the standard. 
 
 
J. CONTAMINATION 
 
It is critical to ensure that subjects who are randomized to receive the intervention formula, are not 
exposed to CM protein during the intervention period.  The risk is especially high in the immediate 
postpartum period when a newborn infant may be given the wrong formula for supplementation in the 
hospital nursery.  To prevent such contamination, infants who are in the study are clearly identified to all 
nursery personnel.  Any formula supplementation for these infants is only with Nutramigen if formula is 
required prior to randomization (interim formula). 
 
 
K. DATA BASE 
 
The database is held at the Pediatrics Epidemiology Center at the University of South Florida, Tampa, 
FL, headed by Professor Jeffrey Krischer. The web sites were built by the DMU in Tampa with the 
assistance of the TRIGR International Coordination Center in Helsinki.  The DMU has established the 
data transfer system between the laboratories and the database using File Transfer Protocol (FTP) 
technique.  The system has recently been tested. The DMU created a password-based security 
management system for all the users who are authorized in the system and the user information was 
collected and saved in the system.  
 
 
L. STATISTICAL METHODS 
 
A two part hypotheses will be tested. The first hypothesis is that the children in the group fed casein 
hydrolysate will have a decreased occurrence of diabetes-associated antibodies in comparison with the 
control group receiving conventional CM-based formula.  The second hypothesis is that the group 
weaned to a casein hydrolysate formula has a reduced incidence of type 1 diabetes.  
 
The first hypothesis will be studied based on a longitudinal data set consisting of repeated measurements 
of several variables at standard time points.  The variables measured at these time points are potential 
confounding factors (i.e., factors the effects of which have not fully been eliminated by the 
randomization), effect modifying factors (such as possible occurrence of differences in weight and 
height development, which may have the ability to modify the effect of the CM exposure considered) 
 
16 

Protocol – revised April 2015 
 
and the outcome variables (e.g., the status of the diabetes-associated antibodies).  At every time point the 
single antibody variable will be dichotomized as negative or positive, and a summary variable will be 
produced (negative if less than two variables are antibody positive and positive if two or more variables 
are antibody positive).  The exposure to formula vs. placebo will be included in the model as an 
indicator variable.  Age at initiation, duration of breast feeding and/or formula use and cumulative 
formula dose will be included in the model as potential effect modifying and confounding factors.  The 
presence of interactions will be tested and the possible effect of the components considered will be 
estimated. 
 
Two different statistical methods will be used to analyze the hypothesis that weaning to a casein 
hydrolysate decreases the occurrence of diabetes-associated antibodies.  First, generalized linear models 
will be fitted to the data (20).  Also random effect models will be used (21).  These models which 
represent a general approach to the problem of modeling repeated measurements with fairly general 
error structures, can allow for missing observations, serial correlations, time-varying covariates, and 
irregular measurement occasions.  Second, for the description of the data, the association of covariates 
with the risk of an elevated antibody at a given point of time will be assessed using logistic regression 
models  (22).  A rather similar statistical approach was used in the DCCT Study  (23). 
 
The analyses of the second hypothesis will use the time of diagnosis of manifest type 1 diabetes as the 
only outcome measure.  The dataset will thus be similar to that of a cohort study, and the statistical 
analyses will be carried out using the proportional hazards regression model  (24) including the milk 
exposure as the risk factor and potential confounding factors a covariates in the model.  Also time-
dependent covariates can be included to assess the effects of potential modifying factors.  The adjusted 
relative risk of type 1 diabetes between the two groups of milk exposure will be estimated.  Also the 
adjusted incidence of the disease in the two groups can be assessed.  
 
 
M. INTERIM ANALYSIS  
 
An interim analysis of autoantibodies and clinical diabetes will be performed repeatedly by the Data 
Safety and Monitoring Board, beginning 2 years after the last subject is recruited, in order to ensure that 
an unexpectedly large protective effect of the intervention will be detected early.  Although the follow-
up will be continued, the code will be opened and the results published if either of these analyses reach 
sufficient statistical significance, as defined by the Data Safety and Monitoring Board.  
 
 
N. ETHICAL ISSUES 
 
Ethical approval has been obtained at each study center.  Written informed consent is requested from the 
parents and if needed also from the child.  If, during the course of this study, an alternative strategy for 
diabetes prevention is proven to be effective, then we will inform the families of this possibility and 
discuss, and likely offer its use in a rational fashion.  
 
Detailed HLA information will be released by qualified personnel only on the request by the parents.  
 
 
17 

Protocol – revised April 2015 
 
The diabetes associated autoantibody results will be disclosed to the families after each annual visit 
starting from the 6-year visit. The family will be informed by the Study Center as soon as the 
autoantibody results have become available.  
 
 
 O. TRAINING, PROJECT PLANNING AND TIME TABLE  
 
To achieve standardization in the implementation of the study protocol, a detailed training program for 
the study personnel is essential.  The national coordinators, nurse and nutrition coordinators participate 
at vital training sessions in their respective regions.  We will utilize the experience gained during the 
second pilot study in training and informing the national investigators and their staff, when we expanded 
the study to Sweden, Estonia and Hungary (investigators’ meetings, site visits by project staff etc.).  
Additional training is provided by the Monitors and other clinical team mentors to their counterparts 
during their visits to the centers. 
 
1. year  
-  Investigator meeting at the beginning of this period to review the recruitment strategies and protocol  
-  Training sessions are organized for study personnel 
-  Recruitment and genetic screening are started 
-  Clinical follow-up of the children and collection of the follow-up samples are initiated 
-  Dietary advice, assessment and maintenance of dietary compliance of families start 
-  Monitoring of the implementation of the dietary intervention and the clinical follow-up 
-  Investigators meeting in the middle of this period to review the start of the project, genetic screening, 
recruitment and participation rate, subject allocation (randomization), study formula distribution, 
implementation of intervention, study baseline assessments (e.g. case report forms), start of the blood 
specimen collection and laboratory functions, avoidance of contamination, protocol adherence 
(compliance), ethical aspects, collaboration in the network 
- Investigators meeting at the end of this period to evaluate the same items as above, and follow-up 
assessments of case report forms and the suitability of dietary advisory material 
2. year  
- Recruitment and genetic screening are continued 
- Clinical follow-up of the children and collection of the follow-up samples are continued 
- Dietary advice and assessment continue 
- Monitoring of the implementation of the dietary intervention and the clinical follow-up continues 
- Investigators meeting in the middle of this period, review of items as listed above for the first  year 
-  Investigators meeting at the end of this period  to evaluate the progress of the project 
3. year  
- Recruitment and genetic screening are continued  
-  Clinical follow-up of the children and collection of the follow-up samples are continued 
 
-  Dietary advice and assessment continue 
-  Monitoring of the implementation of the dietary intervention and the clinical follow-up continues 
-  Analysis of the dietary data from the intervention period 
-  Investigators meeting at the end of this period to evaluate the progress of the project 
4. year  
- Recruitment and genetic screening are continued 
-  Clinical follow-up of the children and collection of the follow-up samples are continued 
 
18 

Protocol – revised April 2015 
 
-  Start of the assays of immunological markers 
- Dietary advice and assessment continue 
-  Monitoring of the implementation of the dietary intervention and the clinical follow-up continues 
-  Analysis of the dietary data are continued 
-  Investigators meeting at the end of this period to evaluate the progress of the study  
5. year  
-  Clinical follow-up of the children and collection of the follow-up samples are continued 
-  Continuation of the assays of immunological markers 
- Dietary advice and assessment continue 
-  Monitoring of the implementation of the dietary intervention and the clinical follow-up continues 
-  Analysis of the dietary data are continued 
- Investigators meeting at the end of the 5. year to evaluate the progress of the study  
6. year  
- Recruitment is completed 
- Clinical follow-up of the children and collection of the follow-up samples are continued 
-  Continuation of the assays of immunological markers 
- Dietary advice and assessment, and monitoring of the implementation of the dietary intervention are 
completed 
- Monitoring of the implementation of the clinical follow-up continues 
-  Analysis of the dietary data are continued 
- Investigators meeting to evaluate the progress of the study  
7. year  
- Clinical follow-up of the children and collection of the follow-up samples are continued 
- Monitoring of the implementation of the clinical follow-up continues 
-  Continuation of the assays of immunological markers 
-  Analysis of the dietary data are continued 
- Investigators meeting to evaluate the progress of the study  
8.- 11. year  
- Clinical follow-up of the children and collection of the follow-up samples are continued 
- Monitoring of the implementation of the clinical follow-up continues 
-  Continuation of the assays of immunological markers 
- Investigators meeting to evaluate the progress of the study  
12. year  
- The last recruited child reaches the age of 6 years 
- Interim analysis of autoantibodies and clinical diabetes is performed 
- Clinical follow-up of the children and collection of the follow-up samples are continued 
- Monitoring of the implementation of the clinical follow-up continues 
-  Continuation of the assays of immunological markers 
- Investigators meeting to evaluate the progress of the study  
13.- 15. year  
- Clinical follow-up of the children and collection of the follow-up samples are continued 
-  Monitoring of the implementation of the clinical follow-up continues 
-  Continuation of the assays of immunological markers 
- Investigators meeting to evaluate the progress of the study  
16. year 
- The last recruited child reaches the age of 10 years 
- Clinical follow-up of the children and collection of the follow-up samples are completed 
 
19 

Protocol – revised April 2015 
 
- Monitoring of the implementation of the clinical follow-up is completed 
-  The randomization code is opened 
17. year  
-  The assays of immunological markers are completed 
- Final analysis of autoantibodies and clinical diabetes is performed 
- Investigators meeting at the end of the study  
 
 
P. PUBLICATION POLICY 
 
The publication policy follows the guidelines published in New England Journal of Medicine (25) and 
the TRIGR Publication and Presentation document.  
 
Q. ORGANIZATION AND ADMINISTRATION OF TRIGR 
 
The trial will be executed by a multinational consortium of clinical research groups, in two large 
regional organizations.  The scale of the study is dictated by the logistics of recruitment, the necessity of 
completion within a reasonable period of time, and the need for ethnic diversity in the study populations.  
Based on the successful development and execution of TRIGR pilot studies, the central coordinating 
center with the study PI will continue to be located in Helsinki (Dr. M. Knip and his deputy, Dr. H.K. 
Åkerblom (PI until July 1, 2008).  The two major regional groups will be: 1. Europe, with a coordinating 
center in Helsinki; 2. North America with a coordinating center in Pittsburgh, PA (Dr. D. Becker) and a 
co-coordinating center in London, Ont. (Dr. J. Dupré).  Each of these regional groups is multicentered, 
as described in Appendix 1.  The trial is nationwide in Finland, Germany, the Netherlands and Canada.  
Satellite centers with investigators who have participated in the development of TRIGR, in New South 
Wales, Australia, will be 'attached' to the European group.  The central major organization functions of 
the study will be carried out in Helsinki and Tampa. These functions include data acquisition, processing 
and storage, randomization and protocol administration.  Monitoring and budgetary administration will 
be carried out by the three major regional coordinating centers. 
 
The essential laboratory functions required for TRIGR will be conducted in three central internationally 
recognized study laboratories 1. for determination of diabetes-related autoantibodies (Dr. M. Knip, 
Helsinki), and 2. for determination of compliance through measurements of CM antibodies (Dr. E. 
Savilahti, Helsinki)  3. MHC typing with optimized turnaround time for Europe will involve the 
laboratory in Turku, Finland (Dr. J. Ilonen).  These laboratories served the same functions in the human 
pilot studies.  An experienced MHC laboratory in Pittsburgh, PA (Dr. M. Trucco) has been added to 
serve the North American centers with rapid turnaround time.  Pilot experiments have been performed to 
optimize tissue typing for the satellites.  Dr. S.M. Virtanen will be responsible for the analysis of dietary 
data.  
 
Three regional fiscal units in Helsinki (Europe), London (Canada), and Pittsburgh (USA) will be 
responsible for operations in these respective regions, and for acquisition and transfer of data to the 
DMU in Tampa, Florida.  However, for governance purposes North America and Europe constitute two 
governance regions.  Communication among participants in each region will be largely by electronic 
means, but group meetings will be essential during the initiation of the study, and throughout the 
recruitment and treatment phase.  Special provision will be required for communication among, and 
support of, the trial coordinators modeled after the DCCT and its epidemiological follow-up (EDIC). 
 
20 

Protocol – revised April 2015 
 
 
Committees 
The two Regional Executive Committees will maintain effective electronic communication among the 
regions, with personal meetings of representatives as needed, through the TRIGR International 
Executive Committee (IEC). This coordinating committee will be under the Chairmanship of the lead 
principal investigator or his delegate, with the co-chairmanship of the national coordinators in Canada 
and United States or their delegates, and with representation of the several constituencies in the study 
groups.   
 
The Data Safety and Monitoring Board supervises the safety issues in the study, and performs the 
interim analyses, described above.  It also supervises compliance issues, like the interpretation of CM 
antibody assays.   
 
Communication 
During randomization and recruitment, rapid communication between each center, the genetics 
laboratories and the central randomization center is essential.  This will be achieved by the development 
of an internet website with Email and fax backup.  Once established, this communication format will be 
used throughout the study. 
 
Participating centers 
The study group members represent a versatile and high-standard expertise in the fields of pediatrics, 
pediatric diabetology, infant nutrition, pediatric gastroenterology, neonatology, obstetrics, nutrition 
science, genetics, immunology and epidemiology.  Many members are internationally recognized 
experts in their fields related to research on type 1 diabetes, particularly on the etiology, pathogenesis 
and prevention of the disease, and they have published extensively on the prediction, prevention and 
etiology of type 1 diabetes.  In addition, most centers in all regions have had substantial clinical trial 
experience in diabetes. 
 
R. ANCILLARY STUDIES OUTSIDE THE CORE PROTOCOL 
 
The present core protocol deals with type 1 diabetes associated autoantibodies and/or manifest type 1 
diabetes.  In addition we intend, with support from other sources (e.g. JDRF) carry out in certain centers 
mechanistic and other ancillary studies approved by the TRIGR Ancillary Studies Committee.  
 
REFERENCES 
 
1.  Kimpimäki T, Kulmala P, Savola K, Vähäsalo P, Reijonen H, Ilonen J, Åkerblom HK, Knip M, the 
Childhood Diabetes in Finland Study Group. Disease-associated autoantibodies as surrogate 
markers of type 1 diabetes in young children at increased genetic risk. J. Clin. Endocrinol. Metab.  
2000: 85: 1126-1132. 
2. Schenker M, Hummel M, Ferber K, Walter M, Keller E, Albert ED, Janka H-U, Kastendiek C, 
Sorger M, Louwen F, Ziegler A-G.  Early expression and high prevalence of islet autoantibodies for 
DR3/DR4 heterozygous and DR4/4 homozygous offspring of parents with Type I diabetes: The 
German BABYDIAB study. Diabetologia 1999: 42: 671-677. 
 
21 

Protocol – revised April 2015 
 
3. Rewers M, Eisenbarth GS, Elsey C, Norris J, Erlich HA, Bugawan T, Klingensmith G.  Target 
population for prevention trials of beta-cell autoimmunity in early childhood.  Diabetologia 1998: 
31: Suppl 1: A86 (abstract).  
4. Veijola R, Reijonen H, Vähäsalo P, Sabbah E, Kulmala P, Ilonen J,Åkerblom HK, Knip M. The 
Childhood Diabetes in Finland (DiMe) Study Group.  HLA-DQB1 defined genetic susceptibility, 
beta-cell autoimmunity and metabolic characteristics in familial and nonfamilial insulin-dependent 
diabetes mellitus. J. Clin. Inv. 1996: 98: 2489-2495. 
5. Lipton RB, Kocova M, LaPorte RE, Dorman JS, Orchard TJ, Riley WJ, Drash AL, Becker DJ, 
Trucco M. Autoimmunity and genetics contribute to the risk of insulin-dependent diabetes mellitus 
in families - islet cell antibodies and HLA DQ heterodimers. Am. J. Epidemiol. 1992: 136:503-512. 
6. Rønningen KS, Spurkland  A, Tait BD, Drummond B, Lopez-Larrea C, Baranda FS, Menendez-
Diaz MJ, Caillat-Zucman S, Beaurain G, Garchon H-J, Ilonen J, Reijonen H, Knip M, Boehm BO, 
Rosak C, Löliger C, Kühnl P, Ottenhoff T, Contu L, Carcassi C, Savi M, Zanelli P, Neri TM, 
Hamaguchi K, Kimura A, Dong RP, Chikuba N, Nagataki S, Gorodezky C, Debaz H, Robles C, 
Coimbra HB, Martinho A, Ruas MA, Sachs JA, Garcia-Pachedo M, Biro A, Nikaein A, 
Dombrausky L, Gonwa T, Zmijewski C, Monos D, Kamoun M, Layrisse Z, Magli MC, Balducci P, 
Thorsby E. (B) HLA class II associations in insulin-dependent diabetes mellitus among Blacks, 
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Protocol – revised April 2015 
 
APPENDIX 1 
 
 
TRIGR: 
Participating Countries and Annual Recruitment Prospects 
 
 
 
Country 
 
Number of centers 
 
  Registered/year 
Australia 
  3 
 
 
 
  95 
Canada 
  18 
850 
The Czech Republic 
  7 
 
 
 
  40   
Estonia 
  2 
 
 
 
  30 
Finland 
16 
 
 
 
300 
Germany 
  1 
 
 
 
  80 
Hungary 
  1 
 
 
 
  50 
Italy 
  2 
 
 
 
  90  
Luxembourg 
1 
 
 
 
  10 
The Netherlands 
  1   
 
 
 
  60 
Poland 
  5 
 
 
 
120 
Spain 
  3 
 
 
 
  75 
Sweden 
10 
 
 
 
100 
Switzerland 
  1    
 
 
 
  25 
USA 
  6 
 
 
         1,050 
TOTAL 
77 
2,975 
 
 
 
 
24