urine Confirmatory Tests PDF

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Urine Confirmatory Testing

CLINITEST Glucose and other sugarsfound in urine can act as reducing substances. These other sugars (galactose, lactose, fructose, and maltose) can be detected in proceduresutilizing glucose to reduce copper. The Clinitest, also known as the Benedict’s Test, is a copper reduction test. Reducing sugars will reduce cupric ions to cuprous oxide, in the presence of strongly alkaline solutions and heat. The reaction produces a color change of blue through green to orange depending upon the amount of reducing substances present in the urine. A test for reducing substances should be included in the routine urinalysis of all pediatric patients. This will provide for the early detection of those metabolic defects which are characterized by the excretion of reducing sugars such as galactose, which is present in the urine in patients with galactosemia. The Clinitest is a self-heating method for the semiquantitative determination of reducing substances in the urine. The tablet contains copper sulfate, citric acid, sodium hydroxide, and sodium carbonate. When placed in a mixture of water and urine, the tablet is rapidly dissolved by the action of sodium carbonate and citric acid, which act as an effervescent. The sodium hydroxide provides the alkaline medium necessary for the reaction, and the heat required is provided by the reaction of sodium hydroxide with water and citric acid. The reducing substances in the urine then react with the copper sulfate to reduce the cupric ions to cuprous oxide. Clinitest Procedure: 1. Place five drops of urine into a glass test tube. (Do not use plastic tubes) 2. Add 10 drops of water and mix by shaking. 3. Drop one Clinitest tablet into the tube and observe the complete reaction. Do not shake the tube during the reaction or for 15 seconds after the boiling has stopped. Warning: The bottom of the tube will become very hot. 4. At the end of the 15-second waiting period, shake the tube gently and then compare with the color chart that is provided. The test is reported as negative, 1/4 % (or trace), 1/2% (1), 3/4% (2), 1% (3), or 2% (4). During the reaction, if the color should rapidly “passthrough” bright orange to a dark brown or greenish-brown, report the result as being greater than 2%. Clinitest is a very accurate procedure if the manufacturer’s directions are carefully followed. Failure to observe the reaction as it takes place will result in a falsely low reading. The “pass-through” phenomenon can occur so rapidly that it can be missed if not observed closely. If measurement beyond 2% is medically desirable, an alternate two-drop method is available. This method involves adding only 2 drops of urine to 10 drops of water, but a special color chart must be used. The two-drop method will allow for quantitation up to 5% but the “pass-through” phenomenon may still occur when very large concentrations of sugar are present.

ACETEST

Ketones, or ketone bodies, are formed during the catabolism of fatty acids. One of the intermediate products of fatty acid breakdown is acetyl CoA. Acetyl CoA enters the Krebs cycle in the body if fat and carbohydrate degradation are appropriately balanced. The first step in the Krebs cycle is the reaction of acetyl CoA with oxaloacetate to yield citrate. When carbohydrate is not available or is not being properly utilized, all available oxaloacetate will be used to form glucose, and so there will be none available for condensation with acetyl CoA.CoA cannot enter the Krebs cycle; therefore, it is diverted to the formation of ketone bodies: 

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Diacetic Acid / Acetoacetic Acid - is the first of the three ketone bodies to be formed and is the form most detectable by either the dipstick or Acetest tablet. Diacetic acid makes up 20% of the total ketone bodies Acetone - makes up 2% of the ketones produced. It is volatile (meaning that it easily goes to a gas state) and is often removed by the lungs. Acetone can be detected on the dipstick and by Acetest Beta-hydroxybutyric acid - makes up the majority of the ketones formed, but is NOT DETECTED by dipstick or Acetest methodology

Acetoacetic acid and beta-hydroxybutyric acid are normal fuels of respiration and are important sources of energy. In fact, the heart muscle and the renal cortex prefer to use acetoacetate instead of glucose. But glucose is the major fuel of the brain in well-nourished individuals, even though the brain can adapt to utilize acetoacetate in the absence of glucose. The odor of acetone may be detected in the breath of an individual who has a high level of ketones in the blood because acetone is eliminated via the lungs. Normally, small amounts of ketones (2–4 mg/dL) are present in the blood.The relative proportion of each is approximately 20% acetoacetic acid, 2% acetone, and 78% -hydroxybutyric acid.There may, however, be considerable proportional variation among individuals. Acetone is lost into the air if a sample is left standing at room temperature. Therefore, urines should be tested immediately or refrigerated in a closed container until testing. The Acetest tablet contains sodium nitroprusside, glycine, a strong alkaline buffer, and lactose (a color enhancer). Acetest can be used to test urine, serum, plasma, or whole blood. Diacetic acid and acetone react with sodium nitroprusside and glycine in an alkaline medium to form a purple color. Acetest will not react with beta-hydroxybutyric acid. Acetest Procedure: 1. Remove a tablet from the bottle and recap immediately. Place the tablet on a piece of clean, dry white paper. 2. Put one drop of urinedirectly on top of the tablet. 3. Compare the color of the tablet with the color chart at 30 seconds. Results are reported as “small, moderate, or large.” The small color block corresponds to approximately 5–10 mg/dL of diacetic acid, the moderate block is 30–40 mg/dL, and the large block is about 80–100 mg/dL.

ICTOTEST

The Ictotest is a tablet test based on the same diazo reaction as the dipsticks. The Ictotesthas a higher sensitivity than the dipstick, detecting as little as 0.05 mg/dL. Interfering substances have less effect on the Ictotest than in the dipstick reaction. The tablet contains 2,6-dichlorobenzenediazoniuimtetrafluoroborate, sulfosalicylic acid, and sodium bicarbonate. The mats that are used in the procedure are made of an asbestos–cellulose mixture. When the urine is placed on the mat, the absorbent qualities of the mat cause the bilirubin to remain on the outer surface. The sulfosalicylic acid provides the acid environment for the reaction. It also acts with the sodium bicarbonate to provide an effervescence which helps partially dissolve the tablet. The diazonium salt then couples with the bilirubin on the mat, giving a blue or purple reaction product.

Ictotest Procedure: 1. Place a square of the absorbent test mat on a paper towel. Using either side of the mat will produce expected results. 2. Place 10 drops of urine onto the center of the test mat. 3. Shake one Ictotest Reagent Tablet into the bottle cap and transfer the tablet to the center of the moistened mat. Do not handle the tablet with your fingers – used clean gloves. Recap the bottle promptly. 4. Place one drop of water onto the tablet. Wait five seconds then place one more drop onto the tablet so the water runs off the tablet onto the mat. 5. Observe the color of the mat around the tablet at 60 seconds. If a blue or purple color develops, the test is positive. All other colors including pink or red are negative.

SULFOSALICYLIC ACID (SSA) This test is based on turbidity formation as the result of acid precipitation of urine proteins. The 3% sulfosalicylic acid precipitates protein in solution, turning the urine specimen milky. The degree of turbidity is graded from trace to 4+.It is important that any other source of turbidity, such as crystals, mucous, cells, etc, be removed by centrifugation. Therefore this procedure must be performed on the supernatant. Unlike the urine protein test on the dipstick, the SSA reaction will detect globulin and Bence-Jones proteins, in addition to albumin. It is also more sensitive for albumin than the dipstick test. The most accurate test for protein still remains the 24 hour urine test performed in the Chemistry department. Another good alternative to the SSA test is the urine protein to creatinine ratio. This test is often used as a back-up test when:   

Specimen has a strong color that masks the dipstick result Specimen has a strongly alkaline pH that results in false positive / increase in the dipstick result The technician has difficulty deciding between the ‘negative’ and the ‘trace’ or ‘trace’ and ‘1+’ squares on the dipstick.

SSA Procedure:

1. Into a clear glass tube, place 5-10 drops of urine – at least the width of your small finger. 2. Add an equal amount of 3% SSA directly into the tube. 3. Shake the tube gently and read for turbidity immediately. 4. Quantitate as follows:

False Positives:  

X-ray contrast media High concentrations of antibiotics, such as penicillin, sulfonamides, or cephalosporin, Tolbutamide metabolites (oral diabetes drug) or Tolectin (anti-inflammatory)

False Negatives:  

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Highly buffered alkaline urine may neutralize the SSA reagent and lower the result. To resolve, the urine may require acidification to a pH of 5-6 before performing the SSA test. Dilute urine Turbid urine - may mask a positive reaction. Again, best practice is to always usesupernatant from a properly spun urine sample.

REFRACTOMETRY 1Refractometry is used to measure specific gravity which measures the concentration of dissolved particles in a specimen. Refractometry determines the concentration of the dissolved particles by measuring the refractive index – which is the comparison of the velocity of light in air with its velocity in a solution. The concentration of dissolved substances within the solution determines the velocity and angle at which the light beam passes through the solution. Refractometer Procedure: The procedure varies somewhat based on particular equipment, but basically a small drop of urine is placed on a glass prism / plate. A flap holds the urine in place. The refractometer is held up towards an area of natural light while the technician looks through the eyepiece to view a scale. The specific gravity is read from the scale at the point of contrast line (area where a boundary between light and dark) crosses the scale. 1. 2.

Calibrate the refractometer with a small amount of distilled water. Place a 2 -3 drops of water on the prism and secure the cover plate. This will evenly distribute the liquid on the prism. 3. Point the prism end of the refractometer toward a light source and focus the eyepiece until the scale is clearly visible.

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Read the scale value at the point where the dark and light portions meet. The water should give a reading of 1.000. This should be done before you begin testing, and after every ten samples or so to ensure that the calibration remains accurate. 5. Place 2-3 drops of urine on the glass plate and close the flap. Hold the refractometer up towards an area of natural light, look though the eye piece and read the specific gravity level off the scale the point where the contrast line (difference between light and dark areas) crosses the scale....