Exercise No. 4 Special Chemical Examination of Urine Special chemical examination of the urine includes test for ketone, RBC, bilirubin, urobilinogen, nitrite, and leukocyte. These tests are considered special because even though they are significant in determining the condition of a patient, it is seldom ordered by physicians. These tests have always been performed in laboratories with a specific procedure for each analyte. Nowadays, these tests are a breeze to perform because of the availability of the urine reagent test strip, which provides a practical and convenient way of testing these analytes. Materials: Urine container 15 mL urine 10 parameter reagent strips Gauze Timer I. Ketones Ketones are products of fat metabolism. There are three intermediate types of ketone; acetone, acetoacetic acid and b-hydroxybutyric acid. But due to the complete breakdown of fat into carbon dioxide and water molecules, ketones are not normally found in the urine. However, when fat is consumed as a source of energy in place of carbohydrates, ketones may appear in the urine. Principle: Reagent strip for ketone employ the principle of sodium nitroprusside reaction. In this reaction acetoacetic acid in an alkaline medium reacts with sodium nitroprusside to produce a purple color. The test does not detect b-hydroxybutyric acid and only measures acetone when glycine is also present. Reading time: 40 seconds Specificity: Specific for acetoacetic acid Sensitivity: at least 5 – 10 mg/dL acetoacetic acid Clinical Significance: Increased Ketones Diabetes mellitus Severe starvation Anorexia Fever Lactic acidosis Propanol poisoning II. Blood Red blood cells present in the urine in large quantities can be noticed by visual inspection of the urine specimen. A reddish urine specimen may be suspected for the presence of RBCs, but RBCs in the urine can be present in its intact form or it can be hemolyzed. Hematuria produces a cloudy red specimen, while hemoglobinuria appears as a clear red urine specimen. However, presence of little number of RBCs in the urine does not always produce a red discoloration. Microscopic examination can detect the presence of RBC but presence of free hemoglobin produced either by hemolytic disorders or lysis of red cells is not detected. Therefore, chemical tests for hemoglobin provide the most accurate means for determining the presence of blood. Principle: Reagent strip for blood use employs the principle, pseudoperoxidase acticvity of hemoglobin to catalyze a reaction between the hem component of both hemoglobin and myoglobin and the chromogen tetramethylbenzine to produce oxidized chromogen. Reading time: 60 seconds Specificity: intact RBC, hemoglobin and myoglobin Sensitivity: 5 -20 RBCs/mL ; 0.015 – 0.062 mg/dL hemoglobin Note: In the presence of free hemoglobin/myoglobin, uniform color ranging from a negative yellow through green to a strongly positive green-blue appears on the pad. In contrast, intact red blood cells are lysed when they come in contact with the pad, and the liberated hemoglobin produces isolated reaction that results in a speckled pattern on the pad. Clinical Significance: Hematuria Associated with renal or genitourinary tract disorder that results in bleeding. Renal calculi Glomerular disease Tumors Trauma Anticoagulant therapy Strenuous exercise Menstruation Hemoglobinuria Transfusion reaction Hemolytic anemia Severe burns Malaria Strenuous exercise Myoglobinuria Muscular trauma Prolonged coma Convulsions Muscle-wasting disease Extensive exertion Drug abuse III. Bilirubin Bilirubin is the degradation product of hemoglobin. There are two types of bilirubin, the water insoluble, unconjugated bilirubin and the water soluble, conjugated bilirubin. Normally, these two types of bilirubin are not seen in the urine. When bilirubin concentration increases or when there is obstruction in the pathway for bilirubin excretion, then bilirubin may be detected in the urine. Only the water-soluble type of bilirubin can be found in the urine. Principle: Reagent strip for bilirubin employs the diazo reaction principle. Bilirubin combines with 2,4dichloraniline diazonium salt or 2,6-dichlorobenzene-diazonium-tetrafluoroborate in an acid medium to produce an azodye. Reading time: 30 seconds Specificity: Conjugated bilirubin Sensitivity: 0.4 – 0.8 mg/dL bilirubin Clinical Significance: Hepatitis Cirrhosis Other liver disorders Biliary obstruction IV. Urobilinogen Bilirubin does not normally show up in the urine because as it is metabolized into its water-soluble form, it goes from the liver into the bile duct and into the intestine where it is further converted by the intestinal bacteria into urobilinogen stercobilinogen. Some urobilinogen is reabsorbed into the blood circulation from the intestines, as it circulates in the blood it passes through the kidneys and is filtered by the glomerulus. Therefore, a small amount of urobilinogen (<1mg/dL) is normally found in the urine. Principle: Reagent strip for urobilinogen employs the Ehrlich’s Aldehyde Reaction principle, in which urobilinogen reacts with p-dimethylaminobenzaldehyde (Ehrlich Reagent) to produce color reaction. Reading time: 60 seconds Sensitivity: 0.2 mg/dL urobilinogen Note: Urobilinogen should be correlated with urine bilirubin test for proper and correct diagnosis. Clinical Significance: Condition Urine Bilirubin Urine Urobilinogen Bile Duct Obstruction +++ Negative Liver Damage + or ++ Hemolytic Disease Negative +++ V. Nitrite This test is useful in detecting nitrate reducing bacteria. Some of the gram-negative bacteria that commonly cause Urinary Tract Infection have enzymes that converts nitrate present in urine into nitrite. Principle: Reagent strip for nitrite employ the Greiss reaction principle, in which nitrite reacts in an acidic medium with an aromatic amine in order to form diazonium salt that in turn reacts with tetrhydrobenzenequioline to produce an azodye. Reading time: 60 seconds Sensitivity: 0.06 – 0.1 mg/dL nitrite ion Clinical Significance: Urinary Tract Infection Cystitis Pyelonephritis VI. Leukocyte Leukocytes are normally seen in the urine, microscopically. Normal range varies from 0-2 to 0-5 per high power field. Reagent strip test for urinary leukocytes is not made to replace microscopic examination, instead it provides a standardized means for detection of leukocyte. Principle: The reagent strip for leukocyte employs the leukocyte esterase reaction which catalyzes the hydrolysis of an acid ester embedded on the reagent pad to produce and aromatic compound and an acid. The compound then combines with diazonium salt to produce a diazonium salt. Reading time: 120 seconds Sensitivity: 5 – 15 WBC/hpf Clinical Significance: Increased urinary leukocytes are indicators of UTI or infection in the urinary tract or in the kidneys. Note: Procedure: 1. Briefly dip the reagent strip into the urine, making sure all the reagent pads are immersed into the sample. Do not let the strip stick to the sides of the glass tubes. 2. Draw the edges of the strip along the rim of the tube to remove excess urine. 3. Blot the edges on an absorbent pad and let the color reaction fully develop and read after the specified reading time for each test pad. 4. Read the results against the provided color reaction spectrum for each test pad. 5. Record results.
