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Technical Briefs |
a author for correspondence: fax 33-4-77-42-14-89, e-mail frey{at}univ-st-etienne.fr
We previously verified (1) the suitability of slide chemistry (2) with the Vitros 250 or 700 analyzers (formerly Ektachem 250 or 700, Johnson & Johnson) for analysis of urine samples. The sodium, potassium, urea, creatinine, phosphorus, and uric acid measurements, however, required the preparation of sample dilutions, which dramatically reduced analysis speed. The approach was further complicated by the fact that the diluting agent for sodium and potassium was the Urine Electrolyte Diluent (Johnson & Johnson), which could not be used for the other tests, which required dilutions with water.
The present study was therefore carried out to attempt to standardize the dilutions with a single diluent and a single dilution. Because the diffusion of the sample in the spreading layer of the slides is important in slide technology, we tried an aqueous diluent selected with added surfactant. Preliminary studies (not shown) showed the possibility of ion determination with a Beckman diluent (wash solution, ref. 668601) without disturbances of the creatinine measurement. Therefore, to obtain the same surface tension with an aqueous solution of more defined composition, we tried a 0.15 g/L solution of Tween 20 (Merck, ref. 822184). This solution could be stored for 1 week. Because the dilution for sodium and potassium was 1:5, the dilution for urea and creatinine was 1:21, and the dilution for phosphorus and uric acid was 1:11 in the original system, we used a single dilution of 1:8 as a compromise, and then collected the following data.
The data obtained in two laboratories were compared. Laboratory 1 was equipped with a Vitros 250, which diluted samples automatically, whereas laboratory 2 used the Vitros 700 with manual dilutions. Tubes containing 7 mL of diluent were prepared in advance, and 1 mL of urine was added to each just before analysis in the practice of laboratory 2.
Briefly, Vitros slides for Na and K used direct potentiometry with valinomycin for K and monensin for Na (3); urea slides used urease to generate ammonia, which reacted to form a color with merocyanine dye (4); creatinine was hydrolyzed to creatine, which in a reaction cascade, led to production of hydrogen peroxide that was measured by triaryl imidazole leuco-dye (4); slides for phosphorus used the formation of an ammonium-phosphomolybdate complex and its reaction with p-methylaminophenol sulfate (5); and uric acid was determined by the uricase peroxidase method (6). The specific urine calibration was used.
The linear regression between the multiple dilution and single dilution
methods was checked for urea, creatinine, phosphorus, and uric acid. In
the case of sodium and potassium, the slopes were different from 1.0,
and the intercepts were different from 0 (P <0.001).
Corrections were carried out by introduction of slope and intercept
values established with the flame photometry reference method
(Eppendorf apparatus) for calibration of each new batch of slides. The
linearity limits were modified when a single dilution was used, as
shown in Table 1
. These new limits were determined by successive dilutions of samples
with high concentrations of analytes.
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The imprecision was checked by comparison of multiple dilutions and
single dilution with the Vitros 250 (laboratory 1) and Vitros 700
(laboratory 2), using control urine (Biotrol, A 02262). The within-day
CVs of the two methods (10 determinations) in the two laboratories were
1%. The mean values (± SDs) for different control ranges,
respectively, in laboratories 1 and 2 were as follows: Na (127.3
± 1.49 mmol/L; 89.3 ± 0.72 mmol/L), K (72 ± 0.67 mmol/L;
31.5 ± 0.3 mmol/L), urea (279.5 ± 3.19 mmol/L; 166 ±
2.06 mmol/L), creatinine (9.28 ± 0.09 mmol/L; 3.67 ± 0.04
mmol/L), phosphorus (17.91 ± 0.23 mmol/L; 2.65 ± 0.018
mmol/L), and uric acid (2.18 ± 0.02 mmol/L; 0.60 ± 0.006
mmol/L).
The day-to-day CVs (30 determinations in laboratory 1 and 50 determinations in laboratory 2 of control urine) were <2% for all analytes. One determination was carried out each day during a 1-month period in laboratory 1 and a 2-month period in laboratory 2. The freeze-dried control urine was reconstituted each week and stored at 4 °C. Every day, a fresh dilution was made. Laboratories 1 and 2, respectively, obtained the following results with a different lot in each laboratory: Na (122 ± 2.47 mmol/L; 121.6 ± 2.49 mmol/L), K (70.88 ± 1.10 mmol/L; 60.1 ± 0.95 mmol/L), urea (280.1 ± 5.83 mmol/L; 220 ± 2.74 mmol/L), creatinine (9.45 ± 0.20 mmol/L; 7.84 ± 0.09 mmol/L), phosphorus (17.13 ± 0.30 mmol/L; 15.85 ± 0.20 mmol/L), and uric acid (2.17 ± 0.04 mmol/L; 1.94 ± 0.02 mmol/L).
The agreement between the single dilution system and the standard
multiple dilution system was assessed by analyzing patient urines by
the two methods with both automatic (Vitros 250) and manual (Vitros
700) dilutions. Differences from the mean were calculated
(7). Fig. 1
shows the differences between methods for each analyte. The
observed differences were small compared with the reference ranges. It
is therefore clear that a single dilution of urine with aqueous Tween
20 can be used for assaying sodium, potassium, urea, creatinine,
phosphorus, and uric acid without large error. Although this improved
method is not yet endorsed by the manufacturer, it makes urine analysis
on the Vitros apparatus much faster with or without automatic
dilutions.
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Footnotes
Laboratoire de Biochimie, Faculté de Médecine, 15 rue Ambroise Paré, 42023 Saint-Etienne Cedex 2, France
References
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), data points for laboratory 1
(automatic dilutions with Vitro 250); (– – – –), mean and 2-SD
values for laboratory 1; (
), data points for laboratory 2 (manual
dilutions with Vitros 700); (———–), mean and 2-SD values for
laboratory 2.