Quantitative urine test strip reading for leukocyte esterase and hemoglobin peroxidase.

BACKGROUND: Recently, urine test strip readers have become available for automated test strip analysis. We explored the possibilities of the Sysmex UC-3500 automated urine chemistry analyzer based on complementary metal oxide semiconductor (CMOS) sensor technology with regard to accuracy of leukocyte esterase and hemoglobin peroxidase results. We studied the influence of possible confounders on these measurements. METHODS: Reflectance data of leukocyte esterase and hemoglobin peroxidase were measured using CMOS technology on the Sysmex UC-3500 automated urine chemistry analyzer. Analytical performance (imprecision, LOQ) as well as the correlation with white blood cell (WBC) and red blood cell (RBC) counts (Sysmex UF-5000) were studied. Furthermore, the influence of urinary dilution, haptoglobin, pH and ascorbic acid as confounders was determined. RESULTS: Within- and between-run imprecision (reflectance signal) ranged from 1.1% to 3.6% and 0.9% to 4.2% for peroxidase and 0.4% to 2.5% and 0.4% to 3.3% for leukocyte esterase. Good agreement was obtained between the UF-5000 for RBCs and peroxidase reflectance (r=0.843) and for WBCs and leukocyte esterase (r=0.821). Specific esterase activity decreased for WBC counts exceeding 100 cells/µL. Haptoglobin influenced the peroxidase activity, whereas leukocyte esterase and peroxidase activities showed a pH optimum between 5.0 and 6.5. A sigmoidal correlation was observed between urinary osmolality and peroxidase activity. CONCLUSIONS: CMOS technology allows to obtain high quality test strip results for assessing WBC and RBC in urine. Quantitative peroxidase and leukocyte esterase are complementary with flow cytometry and have an added value in urinalysis, which may form a basis for expert system development.

Folates from metabolically engineered rice: a long-term study in rats.

SCOPE: The biological impact of folates from folate rice, a metabolically engineered (biofortified) rice line, rich in folates, was investigated. Its consumption may be helpful to fight folate deficiency. Our objective was to investigate the potential of folate rice to supply the organism with folates and evaluate its biological effectiveness using a rat model. METHODS AND RESULTS: Five groups of 12 Wistar rats were monitored during a 7/12-wk depletion/repletion trial. Animals receiving folate-free diet (0 µg/rat/day) and those additionally receiving wild-type rice (on average 0.11 µg/rat/day) suffered from decreased hematocrit and lower folate concentrations in both plasma and RBCs. This resulted in serious morbidity and even lethality during the trial. In contrast, all animals receiving a daily supplement of folate rice or folic acid fortified rice (on average 3.00 µg/rat/day and 3.12 µg/rat/day, respectively) and those receiving a positive control diet (11.4 to 25.0 µg/rat/day), survived. In these groups, the hematocrit normalized, plasma and RBC folate concentrations increased and pronounced hyperhomocysteinemia was countered. CONCLUSION: Using an animal model, we demonstrated that biofortified folate rice is a valuable source of dietary folate, as evidenced by folate determination in plasma and RBCs, the alleviation of anemia and counteraction of pronounced hyperhomocysteinemia.

Potassium-based algorithm allows correction for the hematocrit bias in quantitative analysis of caffeine and its major metabolite in dried blood spots.

Although dried blood spot (DBS) sampling is increasingly receiving interest as a potential alternative to traditional blood sampling, the impact of hematocrit (Hct) on DBS results is limiting its final breakthrough in routine bioanalysis. To predict the Hct of a given DBS, potassium (K(+)) proved to be a reliable marker. The aim of this study was to evaluate whether application of an algorithm, based upon predicted Hct or K(+) concentrations as such, allowed correction for the Hct bias. Using validated LC-MS/MS methods, caffeine, chosen as a model compound, was determined in whole blood and corresponding DBS samples with a broad Hct range (0.18-0.47). A reference subset (n = 50) was used to generate an algorithm based on K(+) concentrations in DBS. Application of the developed algorithm on an independent test set (n = 50) alleviated the assay bias, especially at lower Hct values. Before correction, differences between DBS and whole blood concentrations ranged from -29.1 to 21.1%. The mean difference, as obtained by Bland-Altman comparison, was -6.6% (95% confidence interval (CI), -9.7 to -3.4%). After application of the algorithm, differences between corrected and whole blood concentrations lay between -19.9 and 13.9% with a mean difference of -2.1% (95% CI, -4.5 to 0.3%). The same algorithm was applied to a separate compound, paraxanthine, which was determined in 103 samples (Hct range, 0.17-0.47), yielding similar results. In conclusion, a K(+)-based algorithm allows correction for the Hct bias in the quantitative analysis of caffeine and its metabolite paraxanthine.

Prediction of the hematocrit of dried blood spots via potassium measurement on a routine clinical chemistry analyzer.

The potential of dried blood spot (DBS) sampling as an alternative for classical venous sampling is increasingly recognized, with multiple applications in, e.g., therapeutic drug monitoring and toxicology. Although DBS sampling has many advantages, it is associated with several issues, the hematocrit (Hct) issue being the most widely discussed challenge, given its possible strong impact on DBS-based quantitation. Hitherto, no approaches allow Hct prediction from nonvolumetrically applied DBS. Following a simple and rapid extraction protocol, K(+) levels from 3 mm DBS punches were measured via indirect potentiometry, using the Roche Cobas 8000 routine chemistry analyzer. The extracts' K(+) concentrations were used to calculate the approximate Hct of the blood used to generate DBS. A linear calibration line was established, with a Hct range of 0.19 to 0.63 (lower limit of quantification, LLOQ, to upper limit of quantification, ULOQ). The procedure was fully validated; the bias and imprecision of quality controls (QCs) at three Hct levels and at the LLOQ and ULOQ was less than 5 and 12%, respectively. In addition, the influence of storage (pre- and postextraction), volume spotted, and punch homogeneity was evaluated. Application on DBS from patient samples (n = 111), followed by Bland and Altman, Passing and Bablok, and Deming regression analysis, demonstrated a good correlation between the "predicted Hct" and the "actual Hct". After correcting for the observed bias, limits of agreement of ±0.049 were established. Incurred sample reanalysis demonstrated assay reproducibility. In conclusion, potassium levels in extracts from 3 mm DBS punches can be used to get a good prediction of the Hct, one of the most important "unknowns" in DBS analysis.