28 August 2018

Dried spotting as an alternative sampling tool for biomarker profiling

Marie Curie fellowship project

Clinical trials are demanding in terms of logistics and sample collection. The most common sample types are spot or 24 hour urine and blood. Collection and management of these samples requires assistance of educated personnel. Collection also requires materials such as sample containers or tubes, needles as well as time-restrained procedures and freezers for storage.

Biomarkers of food intake are often molecules which are quickly excreted so sampling must be frequent to attain a good coverage of the diet over time. However, frequent sample collection and the number of volunteer visits in the clinic can be very demanding and costly. Less tedious and invasive sampling techniques are therefore required to ease the sampling procedure, decrease the burden to volunteers, and to lower the costs. Such novel sampling must have documented storage stability and reliable results.

A promising approach is spotting samples on a carrier, letting it dry, and then storing it at room temperature until analysis. An extraction procedure is required to convert the dried samples back into liquid form. Dried blood spots (DBS) or urine spots (DUS) can be collected, temporarily stored and transferred by the volunteers themselves in a much more convenient manner than any conventionally collected liquid sample. However, dried spotting still has its intrinsic analytical limitations and much investigation is required for it to become an accepted methodology for quantitative analysis.

DUS has an advantage compared to DBS since DBS analyses may suffer from hematocrit influence and cannot be considered as a fully non-invasive approach due to use of lancet. DUS on the other hand may be highly variable in concentration due to variation in urine volume. For quantitatively accurate analysis, fluctuation in urine volumes must be taken into account and results corrected. However, spotted urine samples represent unknown fractions (analytes concentration-wise) of the total daily urine production and correction is therefore not trivial.

Within the research project Dietary Biomarkers*, we tested the possibility of using cosmetic cotton swab kits produced in-house for collection of DUS instead of spotting them on a filter paper, which has been so far the method of choice in majority of clinical studies. The aim of this research was primarily to understand the potential of using DUS on cosmetic swabs for metabolite profiling and to test the agreement of qualitative and quantitative data obtained by chemical analysis compared with wet 24 hour urine samples.

We evaluated adjustment based on creatinine concentrations in the DUS extract as a method for correction for the unknown volume. Urine swabs were easier to use for subjects than DBS and more straightforward to obtain than DUS on paper cards. DUS provided the possibility of direct extraction while DUS on paper cards usually require punching of discs beforehand. This difference could be particularly important in trials with a high number of collected samples.

It is important to mention that quantitative results for DUS were underestimated for several metabolites compared to regular urine samples. We also examined variance of individual total daily creatinine excretion, which was found to be maximally 16%, indicating that creatinine correction is feasible. Interestingly, DUS samples contained analytes at measurable levels for at least 9 months when stored dried in closed plastic tubes at room temperature.

Our findings suggest that the individual creatinine correction factor could be used in long–term clinical trials for correction of measurements in DUS, provided that muscle mass is unchanged and the level of analytical offset is acceptable for the research purpose. Future work has to be focused on improving the kit design, especially swabs, to provide better extraction efficiency.

*Dietary Biomarkers is an individual Marie Curie fellowship project conducted by postdoc researcher Rastislav Monošík at the University of Copenhagen and funded by the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 655699.