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Abstract INTRODUCTION:
Dihydropyrimidine dehydrogenase (DPD), a key enzyme in pyrimidine metabolism, catalyses the rate-limiting step in the degradation of fluoropyrimidine chemotherapeutics, including 5-fluorouracil (5FU) and its prodrug capecitabine. Approximately 10-30% of treated patients develop grade ≥3 toxicity, with fatal outcomes reported in 0.1-1% of cases. As DPD is responsible for over 80% of 5FU catabolism, reduced enzyme activity constitutes a major risk factor for severe toxicity, including myelosuppression. While complete DPD deficiency is rare, partial deficiencies are more prevalent, affecting an estimated 3-5% of the Caucasian population. In order to avoid major toxic events caused by a reduced enzyme activity, DPD phenotyping, prior to fluoropyrimidine chemotherapy, is an increasingly implemented approach to identify these (partial) deficiencies. The analysis encompasses determination of the endogenous analytes uracil & dihydrouracil, where elevated plasma uracil levels indicate an impaired DPD functionality and require treatment adjustments. However, a major limitation lies in the preanalytical phase, as uracil levels rapidly increase after blood collection. Consequently, deviations from recommended handling conditions (processing within 1 h at room temperature or 4 h at 4°C) may lead to false positive results.
OBJECTIVE:
This study aimed to evaluate whether conventional dried blood spots (DBS) improve preanalytical stability for uracil determination.
METHODS:
A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of uracil, dihydrouracil, and uridine. A streamlined sample preparation protocol was optimized for extraction of 6 mm DBS sub-punches. Fifteen healthy volunteers were included to assess the suitability of DBS for clinical DPD phenotyping. Paired venous and capillary blood samples were collected on three separate days to evaluate inter-day variability. On each collection day, at least three DBS samples were prepared to assess intra-day variability. Venous DBS (vDBS) were generated by spotting 25 µL of blood onto DBS cards, while capillary DBS (cDBS) were obtained via finger prick. Overall, a total of 135 datapoints per matrix were available. Agreement between matrices (vDBS vs. cDBS) and within-matrix variability (inter-spot variation) were evaluated. The preanalytical stability was assessed for up to two weeks at room temperature using vDBS of the same volunteers.
RESULTS:
Uracil concentrations were significantly elevated in all cDBS (Wilcoxon signed-rank p<0.0001), with a median of 219% relative to vDBS. In addition, the inter-spot variation was substantially greater in cDBS (29%) than in vDBS (7%). For dihydrouracil, a small bias of -7% was observed, while uridine concentrations were comparable between matrices, with limited inter-spot variability. Preanalytical stability assessment in vDBS demonstrated minimal changes over time, with median uracil levels of 105% and 107% after 1 and 2 weeks at room temperature relative to vDBS that only dried overnight. No significant changes were observed for dihydrouracil or uridine under the same conditions.
CONCLUSION:
This study revealed that cDBS are unsuitable for uracil determination within the framework DPD phenotyping owing to poor agreement with vDBS and substantial variability. In contrast, DBS prepared from venous blood exhibit enhanced preanalytical stability and may serve as a robust alternative to liquid samples, offering a practical solution for managing preanalytical variables.
REFERENCES:
Vandenbroucke K, Stepman H, Stove C. Capillary dried blood microsampling is not suited for dihydropyrimidine dehydrogenase (DPD) phenotyping. Clinical Chemistry and Laboratory Medicine (CCLM).2026. https://doi.org/10.1515/cclm-2026-0146
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= Discovery stage. (57.21%, 2026)
= Translation stage. (23.38%, 2026)
= Clinically available. (19.40%, 2026)
