Quantitative impact of pre‐analytical process on plasma uracil when testing for dihydropyrimidine dehydrogenase deficiency

Abstract

Aims Determining dihydropyrimidine dehydrogenase (DPD) activity by measuring patient's uracil (U) plasma concentration is mandatory before fluoropyrimidine (FP) administration in France. In this study, we aimed to refine the pre‐analytical recommendations for determining U and dihydrouracil (UH2) concentrations, as they are essential in reliable DPD‐deficiency testing. Methods U and UH2 concentrations were collected from 14 hospital laboratories. Stability in whole blood and plasma after centrifugation, the type of anticoagulant and long‐term plasma storage were evaluated. The variation induced by time and temperature was calculated and compared to an acceptability range of ±20%. Inter‐occasion variability (IOV) of U and UH2 was assessed in 573 patients double sampled for DPD‐deficiency testing. Results Storage of blood samples before centrifugation at room temperature (RT) should not exceed 1 h, whereas cold (+4°C) storage maintains the stability of uracil after 5 hours. For patients correctly double sampled, IOV of U reached 22.4% for U (SD = 17.9%, range = 0–99%). Notably, 17% of them were assigned with a different phenotype (normal or DPD‐deficient) based on the analysis of their two samples. For those having at least one non‐compliant sample, this percentage increased up to 33.8%. The moment of blood collection did not affect the DPD phenotyping result. Conclusion Caution should be taken when interpreting U concentrations if the time before centrifugation exceeds 1 hour at RT, since it rises significantly afterwards. Not respecting the pre‐analytical conditions for DPD phenotyping increases the risk of DPD status misclassification.

Discrepancies between dihydropyrimidine dehydrogenase phenotyping and genotyping: What are the explanatory factors?

“…In view of the predominant hepatic expression of DPD, such an impact of cytolysis on uracilemia is not surprising and could contribute, at least partially, to the observed intra-individual variability of uracilemia. 14,15 Concerning the influence of sex, the interpretation of our results appears to be more complicated. Indeed, being male was identified as an independent factor of increased uracilemia (in accordance with the results of a previous study 26 ), whereas patients with U ≥ 16 μg/L in the absence of a DPYD variant were more often female.…”

“…13 In France, health authorities recommend DPD deficiency screening by measuring uracilemia, with thresholds of 16 and 150 μg/L for partial and complete DPD deficiency, respectively. 5 Additional studies are urgently needed to validate these questionable thresholds and define dose reduction according to uracilemia, as well as to better characterize the variability of uracilemia 14,15 and its determinants. In addition to preanalytical conditions, 15 uracilemia varies according to the circadian rhythm of DPD 16 and nutritional status.…”

“…5 Additional studies are urgently needed to validate these questionable thresholds and define dose reduction according to uracilemia, as well as to better characterize the variability of uracilemia 14,15 and its determinants. In addition to preanalytical conditions, 15 uracilemia varies according to the circadian rhythm of DPD 16 and nutritional status. 17 Liver dysfunction or inflammation, 18,19 two common conditions in cancer patients known to modify drug metabolism, may also contribute to the variability of uracilemia.…”

Response to “Plasma Uracil as a DPD Phenotyping Test: Pre‐analytical Handling Matters”

“…However, there is no consensus for the maximum time of blood storage and several studies have demonstrated consistent increases in uracil concentrations when whole blood is stored at room temperature before centrifugation. [2][3][4] More specifically, average uracil concentrations were found to be increased by 27% after 1 hour, 2 21% after 1.5 hours, 3 and ~25% after 2 hours. 4 Our unpublished data shows an increase of 12.7% after 2 hours in whole blood and at room temperature.…”

“…The Netherlands; 2 Department of Clinical Chemistry, Erasmus Medical Center, Rotterdam, The Netherlands; 3…”