Urinalysis and other Body Fluids Lab - 03 Chemical Urine Exam

ANALYSIS OF URINE AND OTHER BODY FLUIDS LABORATORY 4. CHEMICAL URINE EXAMINATION OUTLINE • • Introduction Chemical Urine Examination o Reagent Strip (10 Parameters) o Benedict’s Test (Glucose) o Sulfosalicylic Acid Precipitation (SSA) Test (Protein) • INTRODUCTION • • Routine chemical examination of urine has changed dramatically since the early days of urine testing, due to the development of the reagent strip method for chemical analysis. Reagent strips currently provide a simple, rapid means for performing medically significant chemical analysis of urine, including pH, protein, glucose, ketones, blood, bilirubin, urobilinogen, nitrite, leukocytes, and specific gravity. Certain variations relating to chemical reactions, sensitivity, specificity, and interfering substances occur among the products and are discussed in the following sections. Reagent strip brands are also specified by instrumentation manufacturers. CHEMICAL URINE EXAMINATION REAGENT STRIP (10 PARAMETERS) Parameters pH Protein Glucose Ketones Blood Bilirubin Urobilinogen Nitrite Leukocyte Specific Gravity Principle Double indicator system Protein error of indicators Double sequential enzymatic reaction Nitroprusside Reaction Pseudoperoxidase activity of Hgb Diazo Reaction Ehrlich's Reaction (M); Diazo Reaction (C) Greiss Reaction Esterase Hydrolysis of Acid Ester Change in pKa of polyelectrolytes MATERIALS • • • • Reagent Strip Test Tube Test Tube Rack Urine Specimen PROCEDURE: (URINALYSIS TESTING USING SIEMENS MULTISTIX 10 SG REAGENT STRIPS ) 1. 2. 3. Collect fresh urine specimen in a clean, dry container. If specimen has been refrigerated, let it come to room temperature before testing. Mix well immediately before testing. Remove one strip from the bottle of strips and replace the cap. Completely immerse reagent areas of the strip in the urine and remove immediately to avoid dissolving out reagents. While removing, run the edge of the strip against the rim of the urine container to remove excess urine. Hold the strip in a horizontal position to prevent possible mixing of chemicals from adjacent reagent areas and/or contaminating the hands with urine. • • • Compare reagent areas to coresponding color chart on the bottle label at the time specified. Hold strip close to color blocks and match carefully. Avoid laying the strip directly on the color chart, as this will result in the urine soiling the chart. PROPER READ TIME IS CRITICAL FOR OPTIMAL RESULTS. READ: o 30 seconds: GLUCOSE & BILIRUBIN o 40 seconds: KETONE o 45 seconds: SPECIFIC GRAVITY o 60 seconds: pH, PROTEIN, UROBILINOGEN, BLOOD & NITRITE o 120 seconds: LEUKOCYTE ESTERASE o The pH and protein areas may also be read immediately or at any time up to 2 minutes after dipping. After dipping the strip,check the pH area. If the color on the pad is not uniform, read the reagent area immediately, comparing the darkest color to the appropriate color chart. All reagent areas except leukocytes may be read between 1 and 2 minutes for identifying negative specimens and for determination of the pH and specific gravity. A positive reaction (small or greater) at less than 2 minutes on the leukocyte test may be regarded as a positive indication of leukocytes in urine. Color changes that occur after 2 minutes are of no diagnostic value. RESULTS OF THE TEST/S BENEDICT'S TEST (GLUCOSE) SULFOSALICYLIC ACID PRECIPITATION (SSA) TEST (PROTEIN) PRINCIPLE • • Benedict’s test is performed by heating the reducing sugar solution with Benedict‘s reagent. The presence of the alkaline sodium carbonate converts the sugar into a strong reducing agent called enediols. During the reaction, enediols decrease the cupric particles (Cu2+) present in Benedict’s reagent to cuprous particles (Cu+) which appear as red copper oxide (Cu2O) which is insoluble in water. When Benedict’s reagent solution and reducing sugars are heated together, the solution changes its color to orangered/ brick red precipitate. The red-colored cuprous oxide is insoluble in water and hence, separate out from the solution. When the concentration of the reducing sugar is high in the solution, then the color becomes more intense (reddish) and the volume of the precipitate increases in the test tube making it clearly visible. MATERIALS • • • • • • Benedict's Reagent Test Tube Test Tube Rack Water Bath Disposable Pipettes Urine Specimen PROCEDURE 1. 2. 3. Place 5mL of Benedict's reagent in a test tube. Add 8 drops of urine, shake, and boil for 2-5 minutes in a boiling water bath. Allow to cool slowly and take note of the result. RESULTS OF THE TEST/S TABLE OF RESULTS FOR BENEDICT’S TEST Negative No change in color Trace Green opacity. No precipitate Positive Green solution with yellow precipitate Positive Green to yellow solution with yellow precipitate +++ Positive Muddy orange solution with yellow precipitate ++++ Positive Orange to brick red precipitate + + ++ PRINCIPLE • Based on the precipitation of urine protein by strong acid, sulfosalicylic acid. MATERIALS • • • • • • • SSA Reagent Test Tube Test Tube Rack Parafilm Disposable Pipettes Centrifuge Urine Specimen PROCEDURE • • • • • Fill 3/4 a pyrex test tube with urine. Centrifuge urine. Add 3mL of 3% SSA reagent to 3mL urine. Mix by inversion & observe for cloudiness. Grade degree of turbidity. RESULTS OF THE TEST/S TABLE OF RESULTS FOR QUALITATIVE PROTEIN TESTS Negative No Cloudiness + Trace Barely visible cloudines + Positive Distinct cloudiness but non-granular against light with urine visible through it ++ Positive Distinct cloudiness, granular against light +++ Positive Heavy cloudiness with distinct flocculi ++++ Positive Dense heavy cloud with large flocculi