The use of freeze-dried blood samples affects the results of a dried blood spot analysis

Dried blood spot (DBS) microsampling has several advantages over venous blood sampling. In a clinical validation study of tacrolimus microsampling it was noted that tacrolimus DBS concentrations ([Tac](DBS)) were systematically higher than tacrolimus whole-blood concentrations ([Tac](WB)). This observation was explored by investigating the effect of using freeze-dried standards (STFD) for [Tac](DBS) measurement. For all experiments, both non-frozen whole-blood samples and whole-blood samples that were frozen and thawed (to simulate freeze-drying) of 10 patients were analyzed. Multiple tacrolimus concentrations were measured: 1) [Tac](WB), 2) [Tac](DBS), where 15 mu L was volumetrically applied to a pre-punched DBS disk, and 3) [Tac](DBS), where 50 mu L was applied before a 6 mm DBS disk was punched from the card. All tacrolimus concentrations were determined independently using STFD and standards made of non-frozen blood spiked with tacrolimus (STSP). In both non-frozen and frozen and thawed whole-blood samples, [Tac](WB) measured with STFD appeared similar to [Tac](WB) measured with STSP (Ratios 1.061 and 1.077, respectively). In non-frozen samples, the median ratio between the [Tac](DBS) measured with STFD, and [Tac](WB) measured with STFD (the reference method), was 1.396. When blood was volumetrically applied to the DBS card (to eliminate the effect of the spreading over the filter paper), this ratio was 1.009. In conclusion, when using DBS microsampling to quantify concentrations of analytes, one should be aware that using the commercially available freeze-dried blood samples for the preparation of standards may affect the spreading of blood on the filter-paper, leading to a systematic error in the results.