New dip-and-read test for determining leukocytes in urine.

A new reagent strip for the determination of leukocytes in urine (LEUKOSTIX; Ames) is described. The test is based on the esterase activity in leukocytes as a marker. Upon contact between the reagent matrix and a urine containing leukocytes, an amino acid ester is hydrolyzed by the esterase to its corresponding alcohol. The free alcohol then couples with a diazonium salt to produce a purple azo dye. The relative concentration of leukocytes in the urine is obtained by visually comparing the strip reaction with a color chart. Performance of the strip was evaluated in a clinical study involving eight different sites and 867 urine specimens. The comparison method was sediment microscopy; specimens containing five cells or more per high-power field were considered to be positive. Sensitivity was 76.3%, specificity 80.8%. Performance was comparable with that of the CHEMSTRIP LN (Boehringer-Mannheim Diagnostics, Inc.) leukocyte test, which we evaluated concurrently.

A control system for monitoring the performance of leukocyte strip tests.

The control system described here is used to monitor the performance of urinary reagent-strip tests for leukocytes. The presence of leukocyte esterase in the urine is used as a marker for leukocytes in urine. The control system is based on sonicated leukocytes, isolated from whole blood. The esterase activity of this sonicate is determined by spectrophotometry with the N-tosyl-L-alanine ester of 5-phenyl-3-hydroxypyrrole as substrate. The assay result is used to determine the amount of sonicate needed to prepare buffered esterase-containing solutions. Such control solutions mimic leukocytic urines and are stable for 6 h at room temperature. The variability of the control system was tested by preparing it five times in a day on five separate days. The overall CV for Leukostix Reagent Strips (Ames Division, Miles Laboratories, Inc.) when tested with these solutions and analyzed with a reflectance spectrophotometer was 8%; for visual readings it was 10%. The overall CV for Chemstrip LN Reagent Strips (Biodynamics, Indianapolis, IN) was 10%.

Interference by ascorbic acid in test systems involving peroxidase. II. Redox-coupled indicator systems.

Ascorbic acid hampers some test systems based on use of peroxidase (EC 1.11.1.7) and redox indicators, by producing a lag time in color development. With reversible indicators, no color development occurs during the ascorbic acid lag time. With oxidatively coupled indicator systems, such as 3-methyl-2-benzothiazolinone hydrazone (MBTH) and a suitable coupler such as chromotropic acid (CTA; 4,5-dihydroxynaphthalene-2,7-disulfonic acid), ascorbic acid diminishes the rate of color development, but does not abolish it. The effect of ascorbic acid strongly depends on the reaction pH as well as the nature of the coupler used. The ascorbate-elicited reduction (or lag) in color development was inversely proportional to the concentrations of MBTH and virtually unaffected by changes in CTA coupler concentration. The rate of color development following the lag was directly proportional to the concentration of MBTH but unaffected by the CTA. These observations suggest that peroxidase with H2O2 catalyzes the oxidation and activation of MBTH to an oxidized species (MBTHox). This species is reduced by ascorbic acid and at the same time couples oxidatively with CTA. Thus, the activity during the ascorbate-induced lag time reflects this competition of ascorbic acid and coupler for MBTHox. This study of peroxidase/ascorbate lag time with the redox coupled indicator system has led to the selection of fast couplers that are highly resistant to interference by ascorbic acid. Suitable resistant couplers (e.g., chromotropic acid, Chicago acid, and H acid) appear to be aromatic ring systems with highly activating substituents and directing toward electrophilic aromatic substitution at the ortho and para positions.