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Letters to the Editor |
Department of Clinical Biochemistry, University Hospital, Ghent, Belgium
aAddress correspondence to this author at: Department of Clinical Biochemistry, University Hospital Ghent, De Pintelaan 185, B 9000 Ghent, Belgium. Fax 32-9-2404985; e-mail Veronique.stove{at}ugent.be.
To the Editor:
Sample handling requirements for uric acid analysis during recombinant uric acid oxidase (rasburicase, Sanofi-Synthelabo) therapy are a matter of concern. Rasburicase catalyzes the oxidation of uric acid to allantoin, which is easily excreted by the kidney. It is indicated for the treatment and prophylaxis of malignancy- or chemotherapy-associated hyperuricemia (1).
For monitoring uricemia in patients receiving rasburicase therapy, the manufacturer recommends keeping blood samples in ice water immediately after collection and during specimen transport until analysis. When samples are maintained at 4 °C, uric acid concentrations are reported to be adequately preserved (2). However, cold inactivation of the enzyme requires a cooling period to bring the collection tube to 4 °C, during which further degradation of uric acid is possible (3). Furthermore, rack-based modern random access analyzers do not allow maintaining the sample temperature at 4 °C. Moreover, in practice, laboratories are often unaware that the patient has received rasburicase because the blood samples were sent at room temperature to the laboratory as part of routine biochemistry testing (2). In view of the rapid uricolysis by rasburicase, the accuracy of uric acid determinations in patients under rasburicase treatment can be questioned under routine conditions.
We studied inactivation of the therapeutic enzyme to preserve uric acid. A blood sample (0.5 mL) was supplemented with 1 mL 8% perchloric acid (PCA) (4). After centrifugation, (900g, 10 min, room temperature) 1 volume of supernatant was neutralized with 1/2 volume tripotassium phosphate 0.7 mol/L (pH = 13). After centrifugation, the supernatant was analyzed. Uric acid analysis was performed by an enzymatic colorimetric assay (Modular P, Roche Diagnostics) with a detection limit of 12 µmol/L, and between-run imprecision (CVs) of 1.2% and 1.3%, at mean concentrations of 297 and 714 µmol/L, respectively. The influence of hematocrit was evaluated by reconstituting plasma and blood cells of a donor in different proportions, resulting in hematocrit values in the range 0.2–0.5. Based on measurements with 3 different donors, the plasma uric acid concentration (y, µmol/L) correlated to the measured uric acid concentration (x, µmol/L) as follows:
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To investigate its in vitro uricolytic activity, rasburicase (concentration: 1.5 mg/L) was added to heparinized blood samples from healthy volunteers (n = 4). An aliquot was treated with PCA, a 2nd aliquot was promptly placed on ice water, and a 3rd aliquot was kept at room temperature. Uric acid analysis was performed at 30-min intervals for 4 h (Table 1
). In untreated samples stored at room temperature (
20 °C), plasma uric acid concentration decreased to <12 µmol/L within 3 h. When untreated samples were stored at 4 °C and, as under routine conditions, centrifuged at room temperature, uricolysis was much less pronounced. However, uric acid values still showed a marked decrease of 30%, thereby exceeding the maximum error budget of 14.8% (5). In PCA treated samples, calculated uric acid values remained stable at room temperature.
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To mimic sampling conditions, heparinized blood samples from 2 volunteers (uric acid concentrations: 321 and 506 µmol/L) were placed in a heating bath at 37 °C before addition of 1.5 mg/L rasburicase. After addition, an aliquot of 0.5 mL was treated with PCA, while the remaining tube was placed in ice water. Residual uric acid concentrations were highest with PCA treatment (at least 95%), while storage in ice water and centrifugation at 4 °C resulted in a higher but stable loss (up to 30%), probably due to degradation during the equilibration time needed for complete enzymatic inactivation. Overall, our findings are in agreement with Lim et al. (1)(2) who demonstrated that the in vitro uricolytic activity of rasburicase is minimized in samples maintained at 4 °C.
We also evaluated a sample of a patient with a hematologic malignancy who was given rasburicase for prophylaxis of hyperuricemia. Following the manufacturer’s instructions, uric acid concentration was 83 µmol/L, compared to 95 µmol/L after immediate PCA treatment, and <12 µmol/L after 1 h storage at room temperature.
In conclusion, sample pretreatment with PCA appears to be a useful tool for monitoring of plasma uric acid concentrations during rasburicase treatment. We recommend supplying plastic tubes containing 2 mL PCA. A 1 mL syringe can be used to add 1 mL whole blood to this tube. Immediately after addition, the tube should be shaken vigorously for 30 seconds before being sent to the laboratory.
Acknowledgments
We thank Sanofi-Synthelabo for providing rasburicase (Fasturtec®).
References
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