Urinalysis

Urinalysis begins with a macroscopic examination of the urine which describes the color and clarity of the urine. Many factors affect urine color including fluid balance, diet, medications and disease. The following table includes a list of the most common causes of abnormal urine coloration. Color Cloudy Brown Pathologic Causes Phosphorus, pyuria, chyluria, lipiduria, hyperoxaluria Bile pigments, myoglobin Food & Drug Causes Diet high in purine-rich foods causing uricosuria Fava beans, Levodopa, metronidazole (Flagyl), nitrofurantoin, anti-malarial drugs Cascara, levodopa, methyldopa, Senna Amitriptyline, indigo, carmine, IV cimetidine (Tagamet), IV promethazine (Phenergan), methylene blue, triamterene (Dyrenium) Phenothiazines, phenazopyridine (Pyridium) Beets, blackberries, rhubarb, Phenolphthalein, rifampin Carrots, Cascara

BrownishBile pigments, melanin, Black methemoglobin Green or Blue Pseudomonas UTI, biliverdin

Orange Red Yellow

Bile pigments Hematuria, hemoglobinuria, myoglobinuria, porphyria Concentrated urine

Urine samples are initially screened with dipsticks. Performing microscopic analysis on only dipstick positive urine samples is cost effective when the patient population being tested has a low incidence of potential disease. Numerous studies have determined that 6 to 20% of patients with urine sediment abnormalities are missed by this testing strategy. However, most of the missed cases are clinically insignificant and are often due to contaminating bacteria multiplying after urine collection. Urine dipsticks are plastic strips with attached reagent pads for pH, protein, glucose, ketone, bilirubin, urobilinogen, blood, nitrite, and leukocyte esterase. The principle and performance of each dipstick test is summarized below. pH The test is based on a double indicator method (methyl red and bromthymol blue) that covers the entire range of urine pH. Colors range from orange through yellow and green to blue. pH should be measured in fresh urine and read quickly. The pH of urine is an indication of the kidney's ability to maintain a normal plasma pH. Metabolism produces acids that are excreted by the lungs and kidneys. The average adult urine

pH varies between 5 and 8. A diet high in protein produces a more acid urine, while a vegetarian diet often produces a pH greater than 6. Heavy bacterial growth may cause an alkaline shift in urine pH by converting urea to ammonia. Pigmented urine can interfere with pH readings. Bacterial contaminants, blood in the urine and contamination by genital secretions can alter urine pH. Protein The protein test is based on a change in color of a pH indicator (e.g. tetrabromophenol blue) in the presence of varying concentrations of protein when the pH is held constant. The reagent pad contains the indicator and a buffer that holds the pH of the pad at approximately 3. Yellow indicates undetectable protein. The color of positive reactions ranges from yellow-green to green to green-blue. The accuracy of this test depends on having urine that is slightly acidic. Dipsticks can detect protein concentrations as low as 5 to 30 mg/dL. Urine protein concentrations are reported as 30, 100, 300, or 2000 mg/dL. This test is optimized to detect albumin and is less sensitive in detecting globulins. Dipsticks do not detect beta-2- microglobulin or immunoglobulin light chains. Standard urine dipsticks are much less sensitive at detecting urine albumin than other assays. Dipsticks do not detect microalbuminuria. Sensitivity Method Dipstick Protein Spectrophotometric Urine Protein Immunoassay for Urine Albumin Typical Detection Limit(mg/dL) 18 6 0.3 (Relative to Urine Dipstick) 1 3X more sensitive 60X more sensitive

Dipstick testing is useful only when urinary protein exceeds 300 to 500 mg/day or albumin exceeds 10 to 20 mg/day. The major cause of a false positive urine protein is a highly alkaline sample. False positive reactions can also be caused by contamination with quaternary ammonium compounds (zepharin, chlorhexidine) used to clean the skin for a clean catch urine. Excessive contact with urine may wash out the buffering system and lead to a false positive result. Confirmatory tests only need to be performed on those urine samples with positive protein and a pH of 7.5 or greater. Proteinuria can have many causes. Postural proteinuria occurs in 3 to 5% of healthy adults and is characterized by the presence of protein in the urine during the day but not the night. Strenuous exercise, fever, and exposure to extreme heat or cold, pregnancy, eclampsia, shock, and CHF cause functional proteinuria. Hematologic malignancies, such as multiple myeloma, may produce excess immunoglobulin that is excreted in the urine. Renal diseases are a common

source of proteinuria. Approximately 25% of urine specimens containing bacteria will have a positive protein reaction as the only positive dipstick reaction. The esterase reagent is sensitive to 15 leukocytes per hpf, but the protein reagent is sensitive to 6 leukocytes per hpf. Glucose The dipstick test is based on a double enzyme method employing glucose oxidase and peroxidase. Color change ranges from green to brown. Small amounts of glucose (< 15 mg/dL) are normally excreted by the kidney, which is below the 75 mg/dL lower limit of detection of dipsticks, Glucose oxidase is specific for glucose and does not react with lactose, galactose, fructose, or reducing metabolites of drugs. Glucose is reported as 100, 250, 500, 1000, or >1000 mg/dL. Urine specific gravity and temperature may affect test reactivity. High urine specific gravity can reduce color development. Urine should be at room temperature before the test is performed to obtain optimum sensitivity. False positive reactions rarely occur, but may be produced by strong oxidizing cleaning agents. Beta lactam antibiotics such as the penicillins, cephalosporins, carbapenems, and monobactams can cause false positive reactions. Massive amounts of ascorbic acid (vitamin C), salicylates or levodopa can decrease the sensitivity of the test. Negative urine samples from pediatric patients under the age of one should be confirmed with a copper reduction method, such as Clinitest, to detect galactose or lactose. Confirmation only needs to be performed once on a patient. Glucosuria usually occurs when the blood glucose level exceeds 180 mg/dL. Glucosuria most commonly occurs in patients with diabetes, infections, myocardial infarction, liver disease, and obesity. Thiazides, corticosteroids, and birth control pills may precipitate glucosuria. Ketones Dipsticks use the nitroprusside reaction to test for acetoacetic acid. They are less sensitive to acetone and do not detect beta-hyroxybutyrate. The typical diabetic patient with ketoacidosis usually excretes 78% beta-hyroxybutyrate, 20% acetoacetate, and 2% acetone. The reaction of acetoacetic acid with nitroprusside results in the development of color ranging from buff pink to shades of purple. Color reactions are categorized as trace, small, moderate and large that correspond to ketone concentrations of 5, 15, 40 to 80 and 80 to 160 mg/dL of urine, respectively. Dipsticks reliably detect ketone concentrations of 40 mg/dL or more, so moderate and large readings do not need to be confirmed. Trace and small readings should be confirmed by using Acetest. The detection level for Acetest tablets is 20 mg/dL. The presence of ketonuria does not signal the need to do further microscopic evaluation. Normally, urine contains < 2 mg/dL of acetoacetic acid, which is not detectable. A healthy individual may have detectable ketones if he/she has been fasting, strenuously exercising, or is pregnant. Ketones are also detected in children consuming high fat diets. Ketonuria is commonly seen in hospitalized patients due to fasting. Ketones are clinically significant only in the presence of urine glucose. Drugs with free sulfhydryl groups such as penicillamine, N-acetylcysteine,

BAL and ACE inhibitors (captopril and enalapril) cause false positive reactions. Ketones are volatile and evaporate from the specimen with time. False negative results can occur with old urine samples. The reagent pads are extremely sensitive to moisture and may become non-reactive after exposure to humid room air for a few hours. Blood The dipstick test for blood is based on the peroxidase-like activity of hemoglobin. Red cells are lysed on contact with the strip, allowing free hemoglobin to catalyze the liberation of oxygen from organic peroxide. Tetramethylbenzidine is oxidized, producing a color change from orange to green-blue. If intact red cells do not lyse, they may produce speckles on the pad. The sensitivity of dipsticks for hemoglobin is 0.015 to 0.062 mg/dL. This concentration corresponds to 5 to 21 RBCs/uL or 1 to 4 RBCs/hpf of concentrated urine sediment. The reference range for RBCs in normal urine is 0-3 RBC/hpf in males and 0-12 RBCs/hpf in females when concentrated urine sediment is examined. This range corresponds to a concentration of 3 to 20 RBCs/uL of urine. Dipstick sensitivity extends into the reference range. Therefore, trace to 1+ reading may be obtained on urine from as many as 3% of healthy individuals. In healthy individuals, fewer than 1000 red cells are excreted in the urine per minute. When 3000 to 4000 red cells are excreted per minute, 2 to 3 red cells will be seen per high power field, indicating microscopic hematuria. Gross hematuria occurs when more than 1 million red cells are excreted per minute. Hematuria can be due to lesions within the GU tract involving the kidneys, ureters, bladder, prostate, or urethra. The most common disorders include cancer, kidney stones, renal disease, urinary tract infection, and benign prostatic hyperplasia. Transient hematuria can result from menstruation, viral illnesses, strenuous exercise, and mild trauma. Anticoagulant therapy and chemotherapy may also cause hematuria. No etiology can be determined in approximately 45% of cases of microscopic hematuria. A positive dipstick test for blood does not tell whether the reaction is due to red cells, red cell casts, hemoglobin casts, or myoglobin. Many conditions can lead to discrepant dipstick and microscopic findings. Any situation that causes red cell hemolysis will give a positive dipstick and negative microscopic result. Urine should be tested shortly after collection because red cell lysis may occur as the sample ages, if the pH is alkaline, or if the specific gravity is 1.010 or less. Bacterially contaminated urine specimens may contain sufficient peroxidase activity to produce a false positive reaction. False positive reactions can also be caused by vegetable peroxidase. False Positive Dipstick Myoglobin Oxidizing agents - bleach, detergent, iodine Bacterial peroxidase Vegetable peroxidase Betadine False Negative Dipstick Dipsticks exposed to air RBCs settle out & urine not mixed Ascorbic acid (high concentration) Formaldelhyde (preservative tablets) High specific gravity Very high protein Urine pH <5.1 High nitrite from UTI

Captopril (Capoten) Bilirubin The bilirubin dipstick test detects conjugated bilirubin and has a sensitivity of 0.5 to 1.0 mg/dL. This test is based on the binding of conjugated bilirubin to diazotized salts fixed in the test pad in a strong acidic environment to produce a colored compound that is various shades of tan or magenta. Positive dipstick tests are confirmed with the Ictotest. Normal adult urine contains about 0.02 mg/dL of bilirubin, which is not detectable by even the most sensitive methods. Confirmation of positive dipstick bilirubin results is most valuable when the urine specimen is pale yellow. Ictotest is a tablet test that uses a similar chemical reaction but a different test environment. Urine is placed on an absorbent test mat that captures substances within the urine. The reagent tablet is then placed on top of the absorbed urine and water is added to the tablet. The water dissolves the solid diazonium salt and acid in the tablet so that they run onto the mat. The reaction of conjugated bilirubin with the diazonium salt in the acid environment results in the formation of a blue ring around the dissolving tablet. The sensitvity of the tablet test is 0.05 to 0.1 mg/dL, which is about 10 times more sensitive than the dipstick test. The tablet test is also more specific than the dipstick test for bilirubin and its primary use is the detection of false positive dipstick reactions. Since the urine is placed on the mat first in the tablet test, abnormal pigments due to medications or blood metabolites can be detected before the chemical reaction ensues. Other interfering substances are washed through the mat and do not come into contact with the diazonium salt. Also, because the reaction product is blue rather than tan or magenta, fewer interpretation problems are encountered. Examples of medications that produce false positive dipstick and negative Ictotest results include rifampin, phenazopyridium (Pyridium), and nonsteroidal antiinflammatory agents (etodolac, mefenamic acid and flufenamic acid). Bilirubin and urobilinogen tests are valuable in detecting hemolysis, hepatic dysfunction, and biliary obstruction. The results of these two tests should be interpreted together. Bilirubin is unstable and rapidly decomposes during exposure to light. False negative reactions are common if urine is not tested shortly after collection. Chlorpromazine (Thorazine) and selenium can produce false negative results. Urobilinogen Most dipsticks use para-dimethylaminobenzaldehyde in a strongly acid medium to test for urobilinogen. A positive reaction produces a pink-red color. Urobilinogen is normally present in urine at concentrations up to 1.0 mg/dL. A result of 2.0 mg/dL represents the transition from normal to abnormal. False positive results can be caused by medications such as paraaminosalicylic acid, antipyrine, chlorpromazine, phenazopyridine, phenothiazine, sulfadiazine, and sulfonamide. High nitrite concentrations can cause false negative reactions. Pigmented urine can interfere with detection of urobilinogen. Conjugated bilirubin is normally excreted into the bowel where bacteria metabolize it to urobilinogen. Urobilinogen is partially reabsorbed from the gut and excreted in the urine. A positive test indicates increased bilirubin delivery to the gut. Hepatitis produces positive urine bilirubin and urobilinogen. Biliary tract obstruction results in positive urine bilirubin but negative urobilinogen. Hemolytic anemia causes negative urine bilirubin and positive

urobilinogen. Disease Healthy Icteric liver disease Biliary obstruction Hemolytic anemia Urobilinogen Normal Increased Absent Increased Bilirubin Negative Positive Positive Negative

Leukocyte Esterase Pyuria (the presence of leukocytes in the urine) can be detected using the leukocyte esterase reagent strip test. The assay is based on the chemical detection of esterases, which are enzymes contained within the azurophilic granules of polymorphonuclear leukocytes. Esterase level is directly proportional to the number of leukocytes present in a urine sample. The basis of the chemical reaction is the hydrolysis of an ester to form an aromatic alcohol and acid. The aromatic compound combines with a diazonium salt to form an azo-dye that changes to purple. Color intensity read at two minutes is proportional to the number of granulocytes in a sample. Positive results are reported semiquantitatively as trace, 1+, 2+, or 3+. The sensitivity for Multistix reagent strips is 5 cells per high power field (hpf) to 15 cells/hpf while Chemstrip reagent strips have a sensitivity of 20 leukocytes per uL of urine. Because of this relative insensitivity, the absence of leukocyte esterase does not rule out urinary tract infection (UTI). A positive esterase reaction indicates inflammation secondary to UTI or renal disease. Esterase activity from either intact or lysed granulocytes can give a positive result. Lysed granulocytes may produce apparent discrepancies between positive dipstick results and negative microscopic examinations. Lymphocytes do not produce a positive reaction. Other sources of esterase such as eosinophils, Trichomonas, or epithelial cells in vaginal fluid may give false positive results. Oxidizing agents such as bleach or colored substances can produce false positives. False negative results can be caused by high concentrations of ascorbic acid (vitamin C), albumin or other proteins (>500mg/dL), glucose (>3000 mg/dL), or ketones. Urine with high specific gravity can cause a false negative reaction because enzyme is not as readily released from crenated white blood cells. These samples should be examined microscopically so as not to miss clinically significant pyuria. WBC clumping may prevent dispersion of leukocyte esterase and cause a false negative result. Outdated or deteriorated dipsticks are another cause of falsenegative results. Doxycycline, gentamicin and some cephalosporins reduce the reactivity of leukocyte esterase and produce false negative results. Conversely, imipenem, meropenem, and clavulanic acid can cause false positive leukocyte esterase reactions. Most studies comparing the sensitivity of nitrite and leukocyte esterase tests compared to urine culture have demonstrated that leukocyte esterase is a more sensitive indicator of UTI than nitrite.

Nitrite The nitrite test is a rapid, indirect method for detecting bacteriuria. The reaction principle is based on bacterial reduction of dietary nitrate, which is normally present in urine, to nitrite, which is not normally present. Nitrite reacts with para-arsanilic acid on the dipstick to form a diazonium compound that reacts with a benoquinoline to form a pink color. Many of the bacteria that cause UTIs have the ability to reduce nitrate, including Escherichia coli, Klebsiella, Pseudomonas, Enterobacter, and Citrobacter. The optimal specimen is a freshly voided, first morning urine that has been retained in the bladder a minimum of 4 hours, permitting adequate time for conversion of nitrate into nitrite by the bacterial enzymes. A positive nitrite test result indicates UTI with significant bacteriuria. Test sensitivity has been standardized to correspond to a urine bacterial count of 100,000 colony forming units/mL (CFU/mL). Color intensity is not proportional to the degree of bacteriuria; results are simply reported as positive or negative. False positive results can be caused by colored substances in the urine (e.g. phenazopyridine) and prolonged specimen storage at room temperature that allows proliferation of contaminating bacteria. If urinalysis cannot be done within two hours after collection, specimens should be refrigerated to prevent bacterial growth. False-negative nitrite results can occur even in the presence of significant bacteriuria due to a number of possible factors. The causative organisms may lack the reductase enzyme needed to convert nitrate to nitrite. For example, both yeast and gram positive bacteria are reductase negative. Malnourished patients and patients receiving intravenous feeding may have insufficient dietary nitrate to promote the chemical reaction. The duration of urine retention in the bladder may be too short (< 4 hours) to facilitate nitrate reduction. Previous antimicrobial therapy may inhibit bacterial metabolism. In the presence of high numbers of bacteria, nitrite may be further reduced to nitrogen, which is not detected. High concentrations of ascorbic acid or urobilinogen can inhibit the chemical reaction. Of course, outdated or deteriorated dipsticks can also yield false-negatives. Microscopic examination of urine sediment or urine culture should be performed, even with negative nitrite, when clinical symptoms suggest UTI. Vitamin C is a strong reducing agent and interferes with a number of dipstick tests. An evaluation of 4379 urinalysis specimens from outpatients in a single laboratory revealed that 23% contained measurable vitamin C. An oral dose of 100 mg of vitamin C caused falsely negative dipstick tests for blood, glucose and leukocyte esterase in urine samples tested within 4 hours of ingestion. Vitamin C consumption is a likely cause of discrepancies between urine dipstick and microscopic analysis. Specific Gravity The specific gravity of a solution is the ratio of the mass per unit volume of the solution to the mass per unit volume of distilled water. It is a relative measure by weight of the amount of dissolved urinary solutes. All urine contains some solutes and will always have a specific gravity higher than pure water (1.000). Normally, an adult should be able to concentrate the urine to a specific gravity of 1.016 to 1.022. A first morning urine with a specific gravity of 1.023 or higher after overnight fluid deprivation indicates normal renal concentrating capacity. The dipstick specific gravity test is based on the apparent pKa change of polyelectrolytes in

relation to ionic concentration. In the presence of an indicator, colors range from deep blue-green in urine of low ionic concentration through green and yellow-green in urines of increasing ionic concentration. Diabetes mellitus is associated with increased urinary volume and elevated specific gravity due to urinary glucose, which increases the solute content. Diabetes insipidus results in a large urinary volume with low specific gravity. Hyposthenuria means a persistently low urine specific gravity of < 1.007. Renal tubular disease is often manifested early by a loss of concentrating capacity of the kidneys; specific gravity is < 1.018. Later in the disease process, the capacity to dilute the urine is lost and the patient can only produce isothenuric urine with a fixed specific gravity of 1.010. The kidneys cannot concentrate urine to a specific gravity of >1.035. Specific gravity readings greater than 1.035 by refractometer, accompanied by normal specific gravity by reagent strips, usually contain higher molecular weight solutes such as glucose, protein, radiopaque contrast media or drugs. Organic iodides in contrast media such as meglumine diatrizoate (Renograffin, Hypaque) may be seen in the urine sediment for a brief time after injection of the dye. The crystals resemble cholesterol crystals. Reference Range Dipstick reference ranges are summarized in the following table: Analysis Specific gravity Blood Ketones Glucose Protein pH Leukocyte esterase Reference Value 1.003 - 1.030 Negative Negative Negative Negative 4.5 - 8.0 Negative If positive, reported as: Number Small, moderate, large Small, moderate, large 100, 250, 500, 1000, >1000 mg/dL 30, 100, 300, >2000 mg/dL Number Positive

Microscopic Exam A microscopic exam is performed if blood, protein, or leukocyte esterase results are abnormal or if a microscopic exam is specifically requested. The urine is centrifuged and examined microscopically for WBC, RBC, crystals, casts, bacteria and yeast. Both dipstick and microscopic exam should be performed for patient populations with a high incidence of genitourinary tract disease. Microscopic urinalysis cannot be completely eliminated because multiple clinically significant findings can only be detected by examining urine sediment directly. For example, a positive dipstick reaction for blood does not distinguish between red cells, hemoglobin, or casts. Likewise, a positive leukocyte esterase reaction does not distinguish between free WBCs that occur in cystitis, from WBC casts that are characteristic of pyelonephritis. Microscopy can detect

several other clinically significant abnormalities that are not detected by dipsticks including renal tubular epithelial cells and casts, fatty casts, oval fat bodies, crystalline casts, and crystals. Microscopic examination is considered normal if all of the following criteria are met:

0 to 6 erythrocytes per high power field 0 to 6 leukocytes per high power field < 3 hyaline or < 1 granular cast per low power field Absence of any other casts Absence of significant crystals (i.e. cystine, leucine, tyrosine)

Specimen Requirement Specimen requirement is 10 mL from a random urine collection. A first morning void (overnight) specimen is preferred because it is more concentrated and can be assumed to have been in the bladder for a number of hours. A dilute specimen is more likely to yield a false negative result. Urine should be tested as soon as possible after collection. Some determinations such as urobilinogen, bilirubin and pH are only valid if obtained on a fresh specimen. Other chemistry results will begin to change within 2 hours at room temperature. Bacterial growth, cellular degradation, precipitation of amorphous material, and increasing pH due to urease producing bacteria adversely affect protein measurements. A urine more than 24 hours old, even if refrigerated should not be tested. If urine has been refrigerated, it should be allowed to return to room temperature before testing. Refrigeration may cause an increase in specific gravity and precipitation of amorphous phosphates and urates. Glucose sensitivity is adversely affected by not testing at room temperature. Advertise | Biography | Terms of Use | Privacy Policy | Site Map | Copyright 2006 - 2009 by ClinLab Navigator, LLC.