After injecting 8 µl (4.5 µg) of sample, the column was washed for 10 min (at 10 µl/min) with 2% B, and the peptides were then eluted (at 10 µl/min) using the following gradient: 2 70% B over 136 min, 70 95% B over 1 min, and 95% B for 15 min. The column was re-equilibrated with 2% B for 30 min prior to subsequent sample loading.
Internal concentrations were previously corrected to take into account the dilution due to subsequent sampling.
In order to investigate the effect of treatment group on owners' responses to questionnaire items, we first looked at the change in response from Time 1 to subsequent sample times.
Care was taken to collect subsequent samples from same locations.
The first sample was collected when the child was approximately 3 years of age, and attempts were made to collect subsequent samples every 6 12 months over a 2-year period.
At very long intervals between sample points, little information is collected about each trajectory, such that, in the extreme case, fixations are observed as changes in frequency from 0 to 1 at subsequent sample points.
Verbal and written instructions were given, and for each collection participants were asked to discard the first urine specimen of the day and to collect all subsequent samples up to and including the first specimen passed the following morning.
Precise parameter values will result in more reliable model simulations, which can be used for interpretation, or can in turn serve to plan subsequent sampling campaigns to further constrain the model parameters.
The rat organs were first freeze-dried to minimize and to facilitate subsequent sample preparation steps.
To collect the subsequent samples, patients were asked to visit the LUMC outpatient clinic for follow-up sample collection at the preferred time points of 3, 6, 9, 12, and 18 months after transplantation (T3 T18).
