Test Principle

Glucose: This test is based on a double sequential enzyme reaction. One enzyme, glucose oxidase, catalyzes the formation of gluconic acid and hydrogen peroxide from the oxidation of glucose. A second enzyme, peroxidase, catalyzes the reaction of hydrogen peroxide with potassium iodide chromogen to oxidize the chromogen to colors ranging from blue-green to greenish-brown through brown and dark brown.

Ketone: This test is based on the reaction of acetoacetic acid with sodium nitroprusside in a strongly basic medium. The colors range from beige or buff-pink color for a Negative reading to pink and pink-purple for a Positive reading.

Specific Gravity: This test is based on the apparent pKa change of certain pretreated polyelectrolytes in relation to the ionic concentration. In the presence of an indicator, the colors range from dark blue or blue-green in urine of low ionic concentration to green and yellow -green in urine of higher ionic concentration.

Blood: This test is based on the pseudoperoxidase action of hemoglobin and erythrocytes which catalyzes the reaction of 3,3™,5,5™-tetramethylbenzidine and buffered organic peroxide. The resulting colors range from orange to yellow -green and dark-green. Very high blood concentration may cause the color development to continue to dark-blue.

pH: This test is based on the well known double pH indicator method, where bromothymol blue and methyl red give distinguishable colors over the pH range of 5-9. The colors range from red-orange to yellow and yellow -green to bluegreen.

Protein: This test is based on the protein error-of-indicator principle. At a constant pH, the development of any green color is due to the presence of protein. Colors range from yellow for a Negative reaction to yellow -green and green to blue-green for a Positive reaction.

Reagents (Based on dried weight at time of impregnation

Glucose: 16.3%w/w glucose oxidase (Aspergillus niger, 1.3IU); 0.6%w/w peroxidase (Horseradish, 3300 IU); 7.0% w/w potassium iodide; 76.1% w/w buffer and non-reactive ingredients.

Ketone: 7.7% w/w sodium nitroprusside balanced with buffer and non-reactive ingredients.

Specific Gravity: 2.8% w/w bromothylmol blue, 69.0%; poly (methyl vinyl ether/maleic anhydride); 28.2% sodium hydroxide.

Blood: 6.6% w/w cumene hydroperoxide; 4.0% w/w 3, 3™, 5, 5™- tetramethylbenzidine; 89.4% w/w buffer and nonreactive ingredients.

pH: 0.2% w/w methyl red; 2.8% w/w bromothymol blue; 97% w/w nonreactive ingredients.

Protein: 0.3% w/w tetrabromophenol blue; 99.7% w/w buffer and nonreactive ingredients.

Warnings and Precautions

Urine Reagent Strips are for in vitro diagnostic use. Do not touch test areas of Urine Reagent Strips.

Storage

Store at room temperature between 15°-30°C (59°-86°F) and out of direct sunlight. Do not use after expiration date.

Recommended Handling Procedures

All unused strips must remain in the original bottle. Transfer to any container may cause reagent strips to deteriorate and become nonreactive. Do not remove desiccant from bottle. Do not open container until ready to use. Opened bottles should be used within 3 months after first opening. Work areas and specimen containers should be free of detergent and other contaminating substances.

Specimen Collection and Preparation

Collect urine in a clean container and test as soon as possible. Do not centrifuge. The use of urine preservatives is not recommended. If testing cannot be performed within one hour after voiding, refrigerate the specimen immediately. Allow refrigerated specimen to return to room temperature before testing.

Test Procedure

1. Remove from the bottle only enough strips for immediate use and replace cap tightly.

2. Completely immerse reagent areas of the strip in fresh, well-mixed urine. Remove the strip immediately to avoid dissolving out the reagent areas.

3. While removing, touch the side of the strip against the rim of the urine container to remove excess urine. Blot the lengthwise edge of the strip on an absorbent paper towel to further remove excess urine and avoid running over (contamination from adjacent reagent pads.)

4. Compare each reagent area to its corresponding color blocks on the color chart and read at the times specified. Proper read time is critical for optimal results.

5. Obtain results by direct color chart comparison.

Note: All reagent areas may be read between 1-2 minutes for screening positive urine from negative urine. Changes in color after 2 minutes are of no diagnostic value.

Quality Control

For best results, performance of reagent strips should be confirmed by testing known negative and positive specimens or controls whenever a new test is performed or whenever a new bottle is first opened. Each laboratory should establish its own goals for adequate standards of performance, and should question handling and testing procedures if these standards are not met.

Results

Results are obtained by direct comparison of the color blocks printed on the bottle label. The color blocks represent nominal values; actual values will vary around the nominal values.

Limitations of Procedure

Comparison to the color chart is dependent on the interpretation of the individual. It is therefore, recommended that all laboratory personnel interpreting the results of these strips be tested for color blindness.

As with all laboratory tests, definitive diagnostic or therapeutic decisions should not be based on any single test result or method.

Glucose: Moderate amounts of ketone bodies (40mg/dL or greater) may decrease color development in urine containing small amounts of glucose (75-125 mg/dl). However, such concentration of ketone simultaneously with such glucose concentration is metabolically improbable in screening. The reactivity of the glucose test decreases as the SG of the urine increases. Reactivity may also vary with temperature.₃

Ketone: Color reaction that could be interpreted as positive may be obtained with urine specimens containing MESNA or large amounts of phenylketones or L-dopa metabolites.₃

Specific Gravity: The chemical nature of the specific gravity test may cause slightly different results from those obtained with the specific gravity methods when elevated amounts of certain urine constituents are present. Highly buffered alkaline urine may cause low readings relative to other methods. Elevated specific gravity readings may be obtained in the presence of moderate quantities (100-750 mg/dl) of protein.

Blood: The sensitivity of the blood test is reduced in urine with high specific gravity and/or high ascorbic acid content. Microbial peroxidase, associated with urinary tract infection may cause false positive reactions.

pH: If proper procedure is not followed and excess urine remains on the strip, a phenomenon known as running over may occur, in which the acid buffer from the protein reagent area run onto the pH area, causing a false lowering in the pH result.

Protein: False positive results may be obtained with highly alkaline urine. Contamination of the urine specimen with quarternary ammonium compounds may also produce false positive results.₄

Expected Values

Glucose: Small amounts of glucose are normally excreted by the kidney.₅ Concentrations as little as 0.1 g/dl glucose, read either at 10 or 30 seconds, may be significantly abnormal if found consistently. At 10 seconds, results should be interpreted qualitatively; for semi-quantitative results, read at 30 seconds only.

Ketone: Normally, no ketones are present in urine. Detectable levels of ketone may occur in urine during physiological stress conditions such as fasting, pregnancy, and frequent strenuous exercise._6-8 In starvation diets, or in other abnormal carbohydrate metabolism situation, ketones appear in the urine in excessively large amounts before serum ketones are elevated.₉

Specific Gravity: Random urine may vary in specific gravity from 1.003-1.040+. Twenty-four hour urine from normal adults with normal diets and normal fluid intake will have a specific gravity of 0.016-1.022.10 In severe renal damage the specific gravity is fixed at 1.010, the value of the glomerular filtrate.

Blood: Any green spots or green color developing on the reagent area within 40 seconds is significant and the urine should be examined further. Blood is frequently, but not invariably found in the urine of menstruating females.

pH: newborn: 5-7 thereafter: 4.5-8 average: 6.3

Protein: In 24-hour urine, 1-14 mg/dl of protein may be excreted by the normal kidney.₄ A color matching any color block greater than trace indicates significant proteinuria. For urine with high specific gravity, the test area may most closely match the trace color block even though only normal concentrations of protein are present. Clinical judgement is needed to evaluate the significance of trace results.

Specific Performance Characteristics

The performance characteristics of Teco Urine Reagent Strips (URS) have been determined both in the laboratory and in clinical tests. Parameters of importance to the user are sensitivity, specificity, accuracy, and precision. Generally, Urine Reagent Strips (URS) have been developed to be specific for the constituent to be measured with the exception of interferences listed above. (See Limitations of Procedure).

For visually read strips, accuracy is a function of the manner in which the color blocks on the bottle label are determined and the discrimination of the human eye in reading the test. Precision is difficult to assess in a test of this type because of the variability of the human eye. It is for this reason that users are encouraged to develop their own standards of performance.

Glucose: This test is specific for glucose; no substances excreted in urine other than glucose is known to give a positive result. The reagent area does not react with lactose, galactose, fructose, or reducing metabolites of drugs; e.g. salicyclates and nalidixic acid. This test may be used to determine whether the reducing substances found in urine is glucose. Approximately 100 mg/dl glucose in urine is detectable.

Ketone: The ketone test area provides semi-quantitative results and reacts with acetoacetic acid in urine. This test does not react with beta-hydroxybutyric acid or acetone. The reagent area detects as little as 5-10 mg/dl acetoacetic acid in urine.

Specific Gravity: The specific gravity test permits determination of urine specific gravity between 1.000 and 1.030. In general, the specific gravity test correlates within 0.005 with values obtained with the reflective index method.

Blood: At the time of reagent manufacture, the test when read as instructed has a sensitivity to free hemoglobin of 0.015 mg/dl or 5-10 intact red blood cells/mL urine. The test is slightly more sensitive to free hemoglobin and myoglobin than to intact erythrocytes.

pH: The pH test area permits quantitative differentiation of pH values to one unit within the range of 5-9. pH reading is not affected by variation in the urinary buffer concentration.

Protein: The test area is more sensitive to albumin than to globulin, hemoglobin, Bence-Jones proteins, and mucoprotein; a negative result does not rule out the presence of these other proteins. The test area is sensitive to 15 mg/dl albumin. Depending on the inherent variability in clinical urine lesser concentration may be detected under certain conditions.

BIBLIOGRAPHY

1. Free, A.H and Free, H.M.: Urinalysis, Critical Discipline of Clinical Science. CRC Crit. Rev. Clin. Lab. Sci. 3(4): 481- 531; (1972).

2. Yoder, J.Adams, E.C., and Free. H.M.: Simultaneous Screening for Urinary Occult Blood, Protein, Glucose, and pH. Amer. J. Med Tech. 31:285; (1965).

3. Tietz, N.W.: Clinical Guide to Laboratory Tests; W.B. Saunders Company, (1976)

4. Burtis, C.A. and Ashwood, E.R.: Tietz Textbook of Clinical Chemistry 2nd Ed. 2205; (1994).

5. Shchersten, B. and Fritz, H.: Subnormal Levels of Glucose in Urine. JAMA 201:129-132; (1967).

6. McGarry, J.D.: Lilly Lecture, 1978: New Perspectives in the Regulation of Ketogenesis. Diabetes 28: 517-523 May, (1978).

7. Williamson, D.H.: Physiological ketoses, or Why Ketone Bodies? Postgrad. Med. J. (June Suppl.): 371-375: (1971).

8. Paterson, P. et al.: Maternal and Fetal Ketone Concentrations in Plasma and Urine. Lancet: 862-865; April 22, (1967).

9. Fraser, J. et al.: Studies with a Simplified Nitroprusside Test for Ketone Bodies in Urine, Serum, Plasma and Milk. Clin. Chem. Acta II: 372-378; (1965)

10. Henry, J.B. et al.: Clinical Diagnosis and Management by Laboratroy Methods, 16th Ed. Philadelphia: Saunders; (1979).