In devising qualitative tests for urine proteins, it is desirable that the test be negative whenever protein is present in normal concentrations, but that the test be positive whenever protein concentration is greater than 20 to 25 mg./100 ml. The test should not be sensitive to mucins and other proteins of nonrenal origin and it should be capable of rough quantitation. A large number of such tests are available. The older tests are based either on precipitating the proteins by heat or by reaction with anionic protein precipitants. A more recent test depends on a change in the color of an indicator in the presence of protein.
Qualitative tests are best done on fresh morning specimens of urine. Such specimens are usually fairly concentrated, thus making possible the detection of proteinuria early in disease. They will also be free of the orthostatic effect discussed previously. If urines must be held for examination at a more convenient time, and if timed specimens are to be examined, they should be refrigerated and examined within 48 hours. Layering with toluene may be used to minimize microbial growth. Bacterially contaminated specimens (pyelonephritis, lower genitourinary tract infections) should be examined only when fresh.
Heat Coagulation Test
The pH of the urine is checked with bromcresol green pH indicator paper, (pH range, 3.8 to 5.5) and if above pH 5.0, acetic acid, 30 per cent (w/v) is added dropwise until the pH is between 4.0 and 4.6. If any turbidity or insoluble matter is present or is formed (mucoproteins may precipitate on adding the acid), it is removed by centrifugation. About 7 to 10 ml. of clear urine are placed in a 15 x 120 mm. test tube and the upper one-third is gently heated over a Bunsen flame until boiling just begins. The heated part is compared in ordinary light with the lower unheated portion of the specimen. Any turbidity, cloudiness, or precipitate demonstrates the presence of protein. The degree of the reaction is customarily graded 0 to ++++. False positive results may occur and are discussed in the section on interpretation of results.
Sulfosalicylic Acid Test
Into a 15 x 120 mm. test tube, place 5 ml. of clarified urine (pH 4.5 to 6.5). Then 0.50 to 0.80 ml. of 20 per cent (w/v) sulfosalicylic acid is added carefully down the sides of the tube to layer underneath the urine. After 1 minute the interface is examined for the presence or absence of turbidity or cloudiness. A barely evident turbidity (5 to 10 mg./100 ml) is graded as +/-, and increasing degrees of turbidity, cloudiness, or precipitation are graded + to ++++. False positives, due to nonspecific precipitation, may be encountered and are discussed in the section on interpretation of results.
The reagent is prepared by dissolving 100 gm. of reagent grade sulfosalicylic acid (2-hydroxybenzoic, 5-sulfonic acid dihydrate) in about 350 ml. of water with the aid of heat. The warm solution is filtered through two layers of filter paper and the total filtrate diluted to 500 ml. when cool. The solution should be crystal clear and colorless. It is discarded when turbidity or darkening develops.
Sulfosalicylic Acid Test
Into a 15 x 120 mm. test tube, place 5 ml. of clarified urine (pH 4.5 to 6.5). Then 0.50 to 0.80 ml. of 20 per cent (w/v) sulfosalicylic acid is added carefully down the sides of the tube to layer underneath the urine. After 1 minute the interface is examined for the presence or absence of turbidity or cloudiness. A barely evident turbidity (5 to 10 mg./100 ml) is graded as +/-, and increasing degrees of turbidity, cloudiness, or precipitation are graded + to ++++. False positives, due to nonspecific precipitation, may be encountered and are discussed in the section on interpretation of results.
The reagent is prepared by dissolving 100 gm. of reagent grade sulfosalicylic acid (2-hydroxybenzoic, 5-sulfonic acid dihydrate) in about 350 ml. of water with the aid of heat. The warm solution is filtered through two layers of filter paper and the total filtrate diluted to 500 ml. when cool. The solution should be crystal clear and colorless. It is discarded when turbidity or darkening develops.
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