Chemical Screening of Urine by Reagent Strip

Which of the following tests included on a urine chemical reagent strip would never be reported out as negative? Urobilinogen Bilirubin Leukocytes Blood Nitrite

Urobilinogen feedback: Urobilinogen is reported as normal, not negative. A normal urobilinogen result is approximately 0.2-1.0 Ehr U/dL.

Nitrite

The nitrite test on the urine reagent strip detects the presence of nitrate-reducing bacteria (ie, gram-negative bacteria). Bladder infections are usually caused by gram-negative bacteria. These bacteria reduce nitrate to nitrite when urine remains in the bladder at least three hours. Gram-positive bacteria do not produce a positive nitrite reaction because they lack the enzyme nitrate reductase that converts urinary nitrate to nitrite. Nitrite is not present in urine under normal circumstances.

To screen for urinary tract infection, the leukocytes test result should be evaluated along with the results from which of these other reagent strip tests?

Nitrite

The ketone component that is measured by the nitroprusside reaction is: Acetoacetic acid Beta-hydroxybutyric acid Acetone

Acetoacetic acid feedback: The ketone component that is measured by the nitroprusside reaction is acetoacetic acid. A positive ketones result is demonstrated as a color change from purple to violet. The intensity of the violet color is proportional to the concentration of ketone bodies in the urine.

Quality Control

Both a normal and an abnormal urine control must be tested with each new lot of chemical reagent strips, and at least every day that patient testing occurs in order to confirm the accuracy of the reagent strips and the automated reader. Some automated readers also require periodic calibration. Follow the manufacturer's instructions for calibration procedure and frequency. Quality control results must be recorded and corrective action must be taken when the results do not fall within the acceptable ranges.

Which of the following procedures should be followed when performing a manual urine reagent strip test? (choose all that apply) More than one answer is correct. Please select all correct answers Compare strip to color chart to check for correlating color. Handle strips at end away from test area. Remove cap of test strip bottle and leave it off until testing is completed.

Compare strip to color chart to check for correlating color. Handle strips at end away from test area. feedback: Compare strip to color chart to check for correlating color and handling strips at the end away from the test area are the two correct answers. It is incorrect to leave the reagent strip bottle cap off until testing is completed as moisture from the air can alter the test pads on the chemical reagent strips.

A positive reagent strip bilirubin test indicates the presence of what form of bilirubin in the urine? Conjugated Unconjugated Both conjugated and unconjugated

Conjugated feedback: A positive reagent strip bilirubin test indicates the presence of conjugated bilirubin in the urine. Unconjugated bilirubin cannot be excreted by the kidneys because it is bound to albumin and is not soluble in water.

False-Positive and False-Negative NItrate Results

False-positive nitrite results may occur when urine specimens have remained at room temperature for an extended period of time, allowing bacterial contaminants to multiply and produce measurable levels of nitrites. Interference from some medications that cause the urine to become red or orange may lead to an incorrect reading of positive for nitrite. False-negative results may occur in urine specimens that did not remain in the bladder for the length of time needed for the bacteria to reduce a measurable quantity of dietary nitrate to nitrite. A false-negative nitrite test can also occur with severe bacterial infections. The bacteria will further reduce the nitrite to nitrogen, which does not react in the test.

Nitrate in urine is reduced to nitrite by: Gram-positive bacteria Gram-negative bacteria Any bacteria

Gram-negative bacteria feedback: Dietary nitrate in urine is reduced to nitrite by gram-negative bacteria. Gram-positive bacteria do not produce a positive nitrite reaction because they lack the enzyme nitrate reductase that converts urinary nitrate to nitrite.

A urine sample is cloudy pink in appearance. The microscopic examination reveals the presence of intact red blood cells (RBCs). What is the term that is used to describe this condition? Haptoglobinuria Hematuria Myoglobinuria Hemoglobinuria

Hematuria feedback: The presence of RBCs in the urine is termed hematuria. Hematuria is associated with various conditions associated with bleeding in the kidneys or urinary tract, among other causes.

Hemoglobinuria may be due to the lysis of red blood cells within the urinary tract. This can be caused by intravascular hemolysis as the hemoglobin is filtered through the glomeruli. In a healthy individual, the hemoglobin molecule attaches to haptoglobin and bypasses the kidney filtration system. When the hemoglobin/haptoglobin system is overwhelmed, hemoglobin passes into the urine. Hemoglobinuria may be associated with:

Hemolytic anemia Severe burns Transfusion reaction Infection Strenuous exercise

Which of the following may cause a false positive bilirubin result on a urine chemical reagent strip?

Large doses of chlorpromazine feedback: A large dose of chlorpromazine, also known as Thorazine, may cause a false positive bilirubin result on a urine chemical reagent strip. This may be caused by a lowered urine pH in the presence of this compound.

Excessive carbohydrate loss that may occur due to vomiting or rapid weight loss may result in the presence of which of following substances not normally contained in the urine? Glucose Bilirubin Ketones Urobilinogen

Ketones feedback: Normal urine specimens usually yield negative results for the presence of ketones. Low levels of ketones may be detected in the urine during conditions of physiological stress such as fasting, rapid weight loss, frequent strenuous exercise or prolonged vomiting. The presence of ketones in these situations is due to either inadequate intake of carbohydrates or increased loss of carbohydrates.

Clinical Significance of Urine Bilirubin

Liver damage or an obstructed bile duct allows conjugated bilirubin to enter the circulation and ultimately to appear in the urine. Patients with clinical jaundice due to hepatitis or cirrhosis will have bilirubinuria. If the jaundice is due to red cell destruction, there is an increase in unconjugated bilirubin, which the kidneys cannot excrete.

Leukocytes Reaction, continued

Lymphocytes do not contain leukocyte esterase and would not produce a positive leukocytes test on the reagent strip. Positive results should be confirmed by performing a microscopic examination of the sediment. Note that white blood cells would not be seen in the sediment if they have lysed; however, the leukocytes test on the reagent strip would still be positive because the test is based on the detection of esterase, which is still present in the urine.

Clinical Significance of Specific Gravity

Measurement of specific gravity provides information regarding a patient's state of hydration or dehydration. It also can be used to determine loss of renal tubular concentrating ability. There are no "abnormal" specific gravity values. This test simply indicates urine concentration.

Specific Gravity Reagent Strip

PRINCIPLE: Change in pKa of certain pretreated electrolytes in relation to ionic strength of urine. Ionic strength is closely related to specific gravity. The more acidic the urine, the higher the SG.

When a patient has a bile duct obstruction, the bilirubin test portion of the reagent strip is:

Positive because conjugated bilirubin is present and is excreted by the kidneys. feedback: When a patient has a bile duct obstruction, the bilirubin test portion of the reagent strip is positive because conjugated bilirubin is present and is excreted by the kidneys. Unconjugated bilirubin, usually caused by red blood cell breakdown, is not able to be excreted from the kidneys, and will not cause a positive urine bilirubin result.

Hematuria is associated with renal or genital disorders in which the bleeding is the result of irritation to the involved organs or some type of trauma. Examples include:

Renal calculi Pyelonephritis Glomerulonephritis Tumors Trauma Exposure to toxic chemicals or drugs Strenuous exercise

How does ion concentration in the urine relate to specific gravity? Specific gravity increases as ionic concentration increases. Specific gravity decreases as ionic concentration increases. There is no relationship between SG and ionic concentration.

Specific gravity increases as ionic concentration increases. FEEDBACK: Urine specific gravity increases as ionic concentration increases. If there were no solutes present, the specific gravity of urine would be 1.000, which is the same as pure water. Since all urine has some solutes present, a urine SG of 1.000 is not really possible. The upper limit of the test pad on a chemical reagent strip, which is typically 1.035, indicates a concentrated urine, one with many solutes with a smaller amount of water

Which of the following statements are TRUE for specific gravity measured by the urine chemical reagent strip method? (Select ALL that apply) More than one answer is correct. Please select all correct answers The urine chemical reagent strip method is based on the relationship between ionic concentration and specific gravity. The urine chemical reagent strip method is based on the fact that light is refracted in proportion to the amount of total solids dissolved in the urine. The urine chemical reagent strip method is not affected by the presence of x-ray contrast media in the urine specimen.

The urine chemical reagent strip method is based on the relationship between ionic concentration and specific gravity. The urine chemical reagent strip method is not affected by the presence of x-ray contrast media in the urine specimen. feedback: There are two correct statements listed in the answer choices, which accurately describe specific gravity measurements by the reagent strip method. These statements are: The urine chemical reagent strip method is based on the relationship between ionic concentration and specific gravity. AND The urine chemical reagent strip method is not affected by the presence of x-ray contrast media in the urine specimen.

pH Analysis: The Urine Specimen

The urine specimen should be freshly voided. Urine is an ideal medium for the proliferation of bacteria due to the large amount of urea present. These bacteria metabolize urea, producing ammonia that causes the urine pH to become more alkaline. If there is a delay before performance of the test, the sample should be refrigerated.

Urine pH: Acidic and Alkaline

Urine pH results must be evaluated in conjunction with a patient's medical condition and clinical history. Factors to be considered include: Respiratory and metabolic status Renal function Crystal or calculi formation Diet

Significantly increased levels of ketones are detected in the urine with which of the following conditions? Choose all that apply. Uncontrolled diabetes Low-impact exercising Starvation

Uncontrolled diabetes Starvation feedback: Significantly increased levels of ketones are detected in the urine in uncontrolled diabetes and starvation, among other conditions where carbohydrate concentration is decreased by a lack of intake or excessive loss. Low-impact exercises would not typically cause ketones to appear in the urine.

Introduction to Urobilinogen

Urobilinogen is a byproduct of hemoglobin breakdown. It is produced in the intestinal tract as a result of the action of bacteria on bilirubin. Almost half of the urobilinogen produced recirculates through the liver and then returns to the intestines through the bile duct. Urobilinogen is then excreted in the feces where it is converted to urobilin by bacterial enzymes. As the urobilinogen circulates in the blood to the liver, a portion of it is diverted to the kidneys and appears as urinary urobilinogen. Up to 1 mg/dL or Ehrlich unit of urobilinogen is present in normal urine. A result of 2.0 mg/dL represents the transition from normal to abnormal levels of urobilinogen and the patient should be evaluated further. It is important to note that the chemical reagent strip cannot determine the absence of urobilinogen, so a negative result is impossible.

When the glucose result on a urine specimen from an infant is negative on the urine reagent strip, it can be assumed that the specimen is also negative for other reducing substances such as galactose. true or false

false

Handling and Storage of Urine Reagent Strips

he following precautions should be observed when handling and storing urine reagent strips: Store strips according to the manufacturer's recommendations. DO NOT expose strips to moisture, volatile fumes, or direct sunlight. Remove only enough strips for immediate use and immediately recap the bottle. Avoid contamination of test strips. Do not touch the test areas with fingers and do not lay test strips directly on the workbench. DO NOT use discolored strips. Compare the color of the unused strip to the negative area on the color chart provided by the company. The color should be similar. Check the expiration date. Re-label the container with a revised expiration date if the manufacturer states a shortened usage period once the container has been opened.

False-Positive and False-Negative Results

A false-positive result for blood on the urine reagent strip can occur if the collection container or reagent strip is contaminated with oxidizing agents, such as hypochlorite (bleach) or if the specimen is contaminated with povidone-iodine, a strong oxidizing agent used in surgical procedures. Microbial peroxide found in association with urinary tract infections may also cause false-positive results. The muscle tissue form of hemoglobin (myoglobin) is a well-known cause of false-positive blood reactions on the reagent strip. When tissue hemoglobin is present, the urine specimen has a clear red appearance. Patients suffering from muscle-wasting disorders or muscular destruction due to trauma, prolonged coma, or convulsions or individuals engaging in extensive exertion may have myoglobin in their urine. Specific tests for myoglobin, such as immunodiffusion techniques or protein electrophoresis, are needed to confirm the presence of this substance in a urine specimen. Captopril can cause a falsely-decreased test result.If the urine specimen is not mixed before testing, blood cells may settle in the sediment and produce a false-negative result for blood.

Clinical Significance of Leukocytes in Urine

A positive leukocytes test is often accompanied by a positive nitrite test. Together, they confirm the presence of a bacterial urinary tract infection.

Confirmatory Testing for Urine Protein

A secondary method, such as the sulfosalicylic acid (SSA) precipitation test, may provide a readable test result with a highly pigmented urine where the reagent strip result is masked.

Overview of Bilirubin

Bilirubin, a product of hemoglobin catabolism, is characterized by its distinctive yellow pigment. Only conjugated bilirubin is excreted in urine. If bilirubin is elevated and is conjugated, it will be detected by the urine reagent strip. The presence of bilirubin in urine in detectable amounts is always abnormal. Unconjugated bilirubin cannot be excreted by the kidneys because it is bound to albumin and is not soluble in water.

Which of the following substances can cause a false-positive result for blood on the urine reagent strip? Ascorbic acid Bleach High levels of nitrite

Bleach feedback: A false-positive test for blood can occur if bleach contaminates the collection container or the reagent strips. Bleach can trigger the color change normally associated with the presence of red blood cells. It is important to follow up with a microscopic analysis of the urine sediment to confirm the result.

Examples of conditions resulting in benign proteinuria include: (Choose ALL correct answers) Multiple myeloma Dehydration Lupus erythematosus Orthostatic proteinuria

Dehydration Orthostatic proteinuria feedback: The presence of protein in the urine does not always indicate renal disease; benign conditions such as dehydration or orthostatic (postural) proteinuria can also produce a positive test for protein. An individual with orthostatic proteinuria will have a positive test for protein after being in an upright position for several hours, but protein will not be present in the urine if the individual has been laying down. Multiple myeloma and lupus erythematosus do not cause benign proteinuria.

Which of the following conditions produce glycosuria (glucose in the urine)? Choose all that apply. Diabetes mellitus Multiple myeloma Impaired tubular reabsorption/advanced kidney failure Preeclampsia

Diabetes mellitus Impaired tubular reabsorption/advanced kidney failure feedback: Diabetes mellitus and impaired tubular reabsorption/advanced kidney failure can cause glycosuria.

False-Positive and False-Negative Ketone Test Results

Drugs containing free-sulfhydryl groups such as captopril (an antihypertensive drug) and D-penicillamine (an antibiotic) can produce false-positive ketone results on the reagent strip. Highly pigmented urine can also produce a false-positive result. Prolonged room temperature storage of the urine specimen can cause a false-negative test result for ketones. Urine specimens should not be stored at room temperature for more than two hours.

Clinical Significance of Nitrite in Urine

Early detection of bacteria is important in order to prevent cystitis from developing into inflammation or infection involving the kidney and renal pelvis. The nitrite test is used to screen individuals who are at risk for developing urinary tract infections, such as diabetics, persons with recurrent infections, or pregnant women. The test is also useful in evaluating the success of antibiotic therapy that is used to treat a bladder infection.

False-Positive and False-Negative Urine Bilirubin Results

False-positive results may occur when patients are on large doses of chlorpromazine, and may occur in the presence of metabolites of phenazopyridine. When these compounds are present, the urine becomes red. Metabolites of etodolac may cause false-positive or atypical results. False-negative bilirubin reagent strip results are often due to testing a specimen that is not fresh. Bilirubin breaks down when exposed to light. Indoxyl sulfate (indican) can produce a yellow orange-to-red color response, which may interfere with the interpretation of a positive or negative reaction. Positive nitrite due to a urinary tract infection may also cause a false-negative result.

Leukocytes Reaction

Granulocytic white blood cells (ie, neutrophils, basophils and eosinophils) contain esterases. The detection of esterase is the basis of the leukocytes test on the urine reagent strip. Leukocyte esterase reacts with the reagents on the reagent strip (indoxy ester derivative and diazonium salt) to produce a purple color. The intensity of the color produced is proportional to the amount of leukocyte esterase that is present. The granulocytes that are most often detected in urine are neutrophils. Their presence indicates a probable urinary tract infection.

Clinical Significance of Glucose in the Urine

In a healthy individual, almost all of the glucose filtered by the renal glomerulus is reabsorbed in the proximal convoluted tubule. The amount of glucose reabsorbed by the proximal tubule is determined by the body's need to maintain a sufficient level of glucose in the blood. If the concentration of blood glucose becomes too high (160-180 mg/dL), the tubules no longer reabsorb glucose, allowing it to pass through into the urine. Conditions in which glucose levels in the urine are above 100 mg/dL and detectable include:diabetes mellitus and other endocrine disordersimpaired tubular reabsorption due to advanced kidney diseasepregnancy - glycosuria developing in the 3rd trimester may be due to latent diabetes mellituscentral nervous system damagepancreatic diseasedisturbances of metabolism such as, burns, infection or fractures

Urine Specimen Transport

In order to ensure proper stability of the specimen these guidelines should be followed: Ensure the transport container lid is secure and leak-resistant (This is especially important if the specimen will be sent to the laboratory through a pneumatic tube system). Utilize urine containers that are made of break-resistant plastic instead of glass. Verify that the specimen has been properly labeled, using at least two forms of positive patient identification (eg, full name and hospital or medical record number or for outpatients, full name and date of birth). Verify that the time the specimen was collected is documented (urinalysis specimens must be analyzed within 2 hours of collection).

Clinical Significance of Urine Protein, continued

Individuals with diabetes mellitus may excrete small amounts of albumin in the urine (microalbumin), which may signal the beginning of reduced glomerular filtration. Stabilizing the blood glucose level at this time may delay progression of diabetic nephropathy. Diabetes (type I or type II) is a leading cause of renal failure. Microvascular damage caused by excessive renal exposure to glucose can lead to diabetic nephropathy. By the time the urine protein level reaches 30 mg/dL, which is necessary for detection by routine reagent strips, damage to the kidneys may have already occurred. Reagent strips are available that use a dye-binding technique rather than the traditional protein-error of indicators principle. These strips are more sensitive and specific for albumin, detecting levels as low as 8 mg/dL. Women in the last month of pregnancy may develop proteinuria as the first sign of impending eclampsia. Eclampsia is the gravest form of toxemia of pregnancy. The presence of protein in this situation must be evaluated by the physician in conjunction with other clinical symptoms. Benign transient proteinuria may be the result of: exposure to cold, strenuous exercise, dehydration, and/or high fever. Benign transient proteinuria may also occur during the acute phase of a severe illness. Patients over the age of 60 have a greater chance of having protein in their urine. Occult malignancies and glomerulonephritis, which occur more frequently in the elderly, may be signaled by the presence of proteinuria. Orthostatic proteinuria is a benign condition seen most often in young adults. The condition may be caused by pressure on the renal nerve. When this condition is suspected, two urine specimens are tested. One specimen is collected upon arising in the morning, and the second is collected several hours later. When this condition is present, the first morning specimen, after the patient has been in a supine position, will be negative for protein. The second specimen, taken after the patient has been upright for several hours, would be positive for protein.

Urine Specimen Collection

Proper collection of urine specimens is important to avoid contamination of the specimen or deterioration of urine constituents. Ensure the urine collection container is clean and the container manufacturer has guaranteed that interfering substances will not leach into the specimen. If the specimen is a voided specimen, it is important to provide patients with instructions for proper collection. Voided specimens that may be provided to the laboratory for testing include: Random specimens: As the name implies, random specimens can be collected at any time. First morning specimen (also known as an eight-hour specimen). The urine is generally more concentrated in this type of specimen because it has been in the bladder for an extended period of time. This may enhance the detection of cellular elements and protein if present. Midstream clean-catch specimens: The patient is instructed to clean the labial area or tip of the penis with an antiseptic wipe. The patient should then begin voiding into the toilet, collect the midstream portion into the sterile container, and then finish voiding into the toilet. This type of collection is necessary when a urine culture is ordered.

The presence of increased levels of protein in the urine may be an early indicator of which of the following conditions? Renal disease Diabetes Hepatitis Heart disease

Renal disease feedback: The detection of protein in the urine may be an early indicator of renal disease (see pg. 21, Clinical Significance of Urine Protein). Normal physiologic criteria allow low molecular weight proteins, primarily albumin, to be filtered through the glomerular capillary wall. Normally, the majority of the filtered albumin is then reabsorbed by the tubules so that in a normal urine, albumin content is below the threshold of detection (less than 15 mg/dL of albumin). Trace protein is considered normal and in most circumstances does not require further analysis. Detection of increased levels of protein on a urine reagent strip is not an early indicator of diabetes. Both type I and type II diabetes mellitus are causes of renal failure. Microvascular damage caused by excessive renal exposure to glucose can lead to diabetic nephropathy. By the time the urine protein level reaches the 30 mg/dL level that is necessary for detection by routine reagent strips, damage to the kidneys may have already occurred. The presence of protein in the urine is also not an early indicator of hepatitis.

Advantages and Limitations of the Chemical Reagent Strip Method for Specific Gravity (SG)

SG measured with the chemical reagent strip method correlates well with gravimetric measurement, and, unlike the gravimetric or refractometer methods, does not need to be corrected for glucose or protein. Cloudy/turbid urines do not need to be clarified before measuring SG with the reagent strip method. It is the recommended method for determining SG if a urine specimen contains x-ray contrast media or plasma expanders. Alkaline urine can affect the indicator system and lower the SG result on the reagent pad (Table 2). If the result is being read visually, it is recommended that .005 be added to the SG result when the pH is alkaline. Most chemical reagent strip readers, however, will automatically adjust the SG reading for pH.

Semi-Automated Instruments

Several manufacturers offer semi-automated instruments (dipstick readers) for reading reagent strips. Use of an instrument removes the subjectivity of visually interpreting color changes on reagent strips and ensures that tests will be read at the correct time. Transcription errors can also be avoided if the instrument is interfaced with the laboratory information system. The technology employed is based on the principle of reflectance, with the amount of light reflected being inversely related to the concentration of substances present. An example of reflectance is the light that is scattered after light strikes an unpolished surface. Since each component on the dipstick produces a different color reaction, the light source for each test must be at the appropriate wavelength. This is accomplished either by using filters or monochromatic light sources. The percent reflectance is determined by dividing the test reflectance by the calibration reflectance and multiplying by 100. Algorithms are used to change the results obtained into a linear relationship with concentration of analyte.

Protein Error of Indicators

Testing for protein in the urine is based on the phenomenon called the "protein error of indicators" (ability of protein to alter the color of some acid-base indicators without altering the pH). In a solution that does not contain protein, tetrabromphenol blue, buffered at a pH of 3, is yellow. However, in the presence of protein, particularly albumin, the color changes to green, then blue, depending upon the concentration. This method is more sensitive to albumin than to globulin, detecting as little as 5 mg albumin/dL urine. Bence Jones protein and mucoprotein are examples of globulin components that are sometimes present in urine, but are not distinguishable by the chemical reagent strip method for urine protein. False-positive results can occur when testing for urine protein. A urine specimen that has remained at room temperature for an extended period of time may produce a false-positive protein result on a reagent strip. A false-positive may also occur in the presence of bacterial contamination, alkaline medication, quaternary ammonium compounds, such as disinfectants or drugs, and with skin cleansers containing chlorhexidine.

Ruling out False-Positive Urine Bilirubin Test Results

The Ictotest® is used by some laboratories to rule out a false-positive bilirubin dipstick result that is caused by urine color interference. The test is performed by adding drops of urine to an absorbent test mat, placing the reagent tablet on the absorbed urine on the mat, and adding water to the tablet, allowing it to spill over the tablet onto the mat. Since the urine is placed on the mat first in the tablet test, this staining is evident before the reaction is performed and can be accounted for. Also, because the reaction product is blue rather than tan or magenta, there are fewer interpretation problems. The Ictotest® employs the same diazotization reaction as the reagent strip, but should not give a false-positive result with colored urines.

Which of the following can cause a false-positive result for ketones? Presence of bacteria The antihypertensive drug captopril Using a freshly collected urine specimen

The antihypertensive drug captopril feedback: The antihypertensive drug captopril may produce a false-positive ketone test. The presence of bacteria does not interfere with the ketone reagent strip test. A fresh specimen is advisable to prevent false-negative reagent strip results.

A patient suspected of a urinary tract infection has a negative nitrite test, but bacteria are present upon microscopic examination. What may have caused this discrepant result? (Choose ALL correct answers) More than one answer is correct. Please select all correct answers The bacteria present are not nitrate-reducers. The urine specimen may have been tested too soon after collection. The urine was not held in the bladder for a sufficient amount of time for nitrate to be reduced to nitrite. The urine was in the bladder for more than 4 hours.

The bacteria present are not nitrate-reducers. The urine was not held in the bladder for a sufficient amount of time for nitrate to be reduced to nitrite. feedback: When a urine sample shows a negative nitrite test, but bacteria are present upon microscopic examination, the false-negative result could be caused by two possibilities listed in the choices above: The bacteria that is present is not a nitrate-reducer OR the urine was in the bladder for an insufficient amount of time for nitrate to be reduced to nitrite.

pH Reaction

The pH pad on the reagent strip contains methyl red and bromothymol blue indicator dyes. The color change that occurs in this test area correlates with the urine's pH. Sensitivity to pH ranges from 5.0 (acid pH with a red-orange color on the reagent strip) to 9.0 (alkaline pH with a blue-green color on the reagent strip). Physiological urine pH ranges from 5.0 to 8.0. Note: The first morning urine sample of a healthy individual usually yields a urine pH of 5.0 to 6.0.

Clinical Significance of Urine Protein

The presence of an increased amount of protein in a urine specimen is often the first indicator of renal disease. Proteinuria may signal severe kidney damage, be a warning of impending kidney involvement, or be transient and unrelated to the renal system. Further quantitative testing of urine for protein may be needed to determine the significance of the proteinuria. Proteinuria related to kidney impairment may be due to glomerular membrane damage caused by toxic agents, immune complexes found in lupus erythematosus, or streptococcal glomerulonephritis. The amount of protein present in urine samples from patients with glomerular damage usually ranges from 10-40 mg/dL. If the urinary protein is due to a disorder that affects tubular reabsorption, the urine protein quantities will be much greater. In patients with multiple myeloma, proteinuria is due to the excretion of the Bence Jones protein. This low molecular weight protein produced by a malignant clone of plasma cells circulates in the blood and is filtered in the kidneys in quantities exceeding the tubular capacity. This excess protein is excreted in the urine.

Bilirubin Reaction

The test for bilirubin on the urine chemical reagent strip is based on the formation of an azobilirubin compound resulting from a reaction of bilirubin in an acid medium with diazotized 2, 4 dichloroaniline. The color of this compound ranges through various shades of tan. Some sources describe the colors produced as shades of tan-to-pink-to-violet. Since other pigments in the urine may influence the test results, this test is more difficult to interpret than other urine reagent strip tests. Colors that are unlike either the positive or negative color blocks on the color chart may be due to the presence of bilirubin-derived bile pigments. Any urine that demonstrates an atypical color on the bilirubin test strip should be tested further. Even a slight change in color should be considered significant since bilirubin is never present in normal urine.

Blood Reaction

The test for blood on the reagent strip is based on the peroxidase-like activity of hemoglobin, which catalyzes the reaction of cumene hydroperoxide and 3, 3', 5, 5' tetramethylbenzidine. The test is sensitive to free hemoglobin, myoglobin, and a minimum of 5 intact RBCs per microliter of urine.

Glucose Reaction

The test for glucose involves a double-sequential enzyme reaction. In the first reaction, glucose oxidase catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide. Then, the peroxidase in the glucose pad catalyzes the oxidation of a chromogen by the hydrogen peroxide to form a colored product. This method is specific for glucose; it does not react with other reducing substances (eg, lactose, fructose, and galactose). NOTE: The urine specimen should be analyzed while at room temperature for these enzyme reactions to occur properly.

Clinical Significance of Urobilinogen in Urine

Urinary urobilinogen may be increased in the presence of a hemolytic process such as hemolytic anemia. It may also be increased with infectious hepatitis, or with cirrhosis. Comparing the urinary bilirubin result with the urobilinogen result may assist in distinguishing between red cell hemolysis, hepatic disease, and biliary obstruction, as shown in the table below: *Urine chemical reagent strip methods cannot distinguish normal urobilinogen from absent urobilinogen, as might be seen in complete biliary obstruction.

Nitrite Reaction

When present, nitrite react with p-arsanilic acid in the reagent pad to produce a diazonium compound. The diazonium compound then combines with 3-hydroxy-1,2,3,4 tetrahydrobenzo-(h)-quinolin to produce a pink color.

Ketones Overview

When the body breaks down fat for energy, three intermediate products are formed. These products, collectively referred to as ketones, include acetone, acetoacetic acid, and beta-hydroxybutyric acid. Normally, the body gets the energy it needs from carbohydrates in the diet. However, stored fat is broken down and ketones are produced and appear in the urine if the diet does not contain enough carbohydrate to supply the body with glucose for energy or if the body cannot use glucose properly.

Follow-up Testing for Positive Urine Protein

A 24-hour urine protein may be ordered if a large amount of protein is detected with the dipstick method or if protein persists in the urine. A 24-hour urine protein may also be ordered if the physician suspects the release into the urine of protein other than albumin.

False-Positive and Falsely-Decreased Results

A false-positive or falsely elevated result may be caused by vaginal contamination of the urine specimen or a highly-colored specimen. Falsely-decreased results may occur in the presence of significantly elevated protein, glucose, or specific gravity (significantly elevated specific gravity can crenate the white blood cells, leaving them unable to release esterases). The drugs cephalexin and gentamicin may also cause a falsely-decreased leukocytes test result.

False-Positive and False-Negative Urobilinogen Results

A false-positive urobilinogen reaction on the reagent strip may occur when substances known to react with Ehrlich reagent are present in the urine, including porphobilinogen*, sulfonamides and p-aminosalicylic acid. Drugs that contain azo dyes, such as Azo Gantrisin®, have a gold color that masks the reaction, causing a false-positive reaction. Atypical color reactions may be obtained in the presence of high concentrations of p-aminobenzoic acid. Due to the instability of urobilinogen, a false-negative result may occur if the urine specimen remains at room temperature and exposed to light for an extended period of time. A false-negative result may also occur if formalin is present. *The urobilinogen reagent strip test is not a reliable method for the detection of porphobilinogen.

Chemical Urinalysis Reagent Strips

A urine reagent strip (dipstick) is a firm plastic strip to which pads saturated with chemicals are affixed. The chemicals in the pads indicate the presence of specific substances in the urine. The strips may also indicate the pH and specific gravity of the urine, depending on the type of strip that is used. Urine reagent strips are available for the detection of glucose, bilirubin, ketones, blood, protein, urobilinogen, nitrite, and leukocytes.

Procedural Precautions

Although the procedure is simple to perform, accurate results depend on careful adherence to manufacturer's directions and adequate quality control. Normal and abnormal controls should be tested whenever a new lot of strips is opened, and at the frequency defined by the laboratory's procedure. If quality control results do not correspond to the published control values, the problem must be resolved before patient samples are tested. Intensely colored urine may make it difficult to correctly interpret color reactions on the dipstick, as illustrated below. The affected tests should not be reported from the dipstick. It would be necessary to use an alternative method of testing if available.

Nitrite Test Sensitivity

Any degree of uniform pink color should be considered positive, suggesting the presence of 105 or more organisms/mL. Detection of low levels of nitrites may be enhanced by placing the activated test strip against a white background. Note that color development is NOT proportional to the number of bacteria present.The test is specific for nitrite and does not react with any other substances normally present in urine. Negative results do not necessarily rule out a urinary tract bacterial infection. An infection may be caused by bacteria that do not reduce nitrate.

Automated Systems

Automation of urinalysis, including urine microscopy, is becoming more commonplace, even in smaller laboratories. These systems currently use reagent strips, as are used for manual or semi-automated methods, for chemical analysis. The urine constituent evaluation on an automated system offers several advantages over manual microscopic sediment examination. A greater number of elements are viewed and evaluated by the instrument than can be viewed and evaluated with a manual sediment examination, providing a more accurate enumeration. Single-particle analysis detects small numbers of constituents that may be missed with a manual microscopic examination. Standardization of the analysis removes the variables that are associated with manual methods. Even though attempts are made to standardize manual methods, they are still manual methods and subject to examiner variations. Automation can improve the accuracy of the count, but test results are still dependent on the quality of the sample that is used. Preanalytic errors, including incorrect identification of the specimen, prolonged room temperature storage of the sample prior to testing, and insufficient mixing of the sample before analysis, impact the accuracy and reliability of test results. Each manufacturer will specify which urine chemical reagent strips are acceptable for use on their instrumentation. Use of an unapproved reagent strip could also cause erroneous results.

Clinical Significance of Urine Ketones

Ketones are usually absent in urine. Their presence most likely indicates that the body is using fats rather than carbohydrates for energy. High levels of ketones may be present in the urine of individuals with uncontrolled diabetes because the body's ability to metabolize carbohydrates is defective. The presence of ketones in the urine is a valuable aid to managing and monitoring individuals with diabetes mellitus. Ketonuria is an indication that the insulin dose needs to be increased. Electrolyte imbalance and dehydration may occur when ketones accumulate in the blood. If these conditions are not corrected by adjusting the dose of insulin, the patient may develop ketoacidosis and ultimately diabetic coma. Low levels of ketones may also be detected in the urine during conditions of physiological stress such as fasting, rapid weight loss, frequent strenuous exercise or prolonged vomiting. The presence of ketones in these situations is due to either inadequate intake of carbohydrates or increased loss of carbohydrates.

False-Positive and False-Negative Urine Glucose Results

No other substance excreted in the urine is known to give a positive result for glucose. False-positive results can be attributed to interfering substances in the environment where the strips are stored or used, such as hydrogen peroxide (H2O2) or bleach (hypochlorite). False-negative results occur when elements present in the urine interfere with either the enzymatic reaction or prevent the oxidation of potassium iodide. Examples of some substances that may produce false-negative results, depending on the reagent strip that is used, include: large quantities of ketones aspirin ascorbic acid > 50 mg/dL levodopa 5-hydroxyindoleacetic acid homogentisic acid sodium fluoride (a preservative) A specific gravity higher than 1.020 may lower glucose reagent sensitivity, especially in the presence of a high (alkaline) urine pH. Exposing reagent strips to excess humidity may also reduce glucose reagent reactivity. NOTE: Check the package insert of the reagent strips used in your laboratory for interfering substances that may affect glucose results.

Introduction to Hematuria

The term hematuria is used to describe the presence of intact red blood cells in the urine. The urine may be cloudy/red or pink in color and red blood cells (RBCs) are visible upon microscopic examination. If RBCs have been destroyed, hemoglobin will be present in the urine. The term, hemoglobinuria, is used to describe this condition. The color of the urine will be pink or red but clear rather than cloudy. The presence of only five RBCs per microliter of urine is considered to be clinically significant. For this reason, a chemical test is needed to detect quantities of blood too small to change the color of the urine. Microscopic examination is used to differentiate between hematuria and hemoglobinuria, if the chemical reagent strip is positive for blood.

Ketone Reaction

The test for ketone bodies is based on a nitroprusside reaction. Acetoacetic acid reacts with sodium nitroferricyanide and glycine in an alkaline medium to produce a violet-to-purple colored complex. The ketone pad on the reagent strip can detect as little as 5 mg/dL acetoacetic acid in urine. It does not react with acetone or B-hydroxybutyric acid.

Urobilinogen Reaction

The test for urobilinogen is based on the Ehrlich Aldehyde Reaction. P-dimethylaminobenzaldehyde in an acid medium with a color enhancer reacts with urobilinogen to form a pink-red color. The urine reagent strip reactivity increases with increasing temperature. The optimum temperature for testing is 22-26°C. A freshly voided sample should be tested to ensure optimal results.

Measuring Specific Gravity (SG)

The urine reagent strip measures specific gravity (SG) in increments of 0.005 with readings from 1.000 to 1.030. The test principle is based on a change in pKa (the negative log of the acid disassociation) of certain pretreated electrolytes (methyl vinyl ether/maleic anhydride) in relation to ionic concentration of the urine. These electrolytes in the specific gravity area contain acid groups that dissociate according to the ionic concentration of the specimen. The more ions in the specimen, the more acid groups will dissociate, releasing hydrogen ions and causing a more acid pH. The reagent area contains a pH indicator (bromothymol blue) which demonstrates the change in pH. The higher the specific gravity of the urine specimen, the more acidic the reagent area will become. The colors of the reagent area will range from deep blue-green in urines of low ionic concentration to green-to-yellow green in urines of increasing ionic concentration, and consequently, higher specific gravity.

Reagent Strip Procedure: Manual or Semi-Automated Methods

Using a fresh, well-mixed uncentrifuged urine, hold the reagent strip by the opposite end from the test areas and dip the stick into the specimen so that all test areas are immersed in the specimen. Remove the stick immediately. Prolonged immersion in the sample may wash out the test reagents. Hold the strip in a horizontal position and run the edge of the strip against the rim of the urine container or touch the long edge of the strip to absorbent towel or gauze to remove excess urine (do not blot the strip). If you are using a dipstick reader, place the strip immediately onto the tray of the reader. Replace the cap on the container to prevent deterioration of remaining strips.