Chapter 5 - Chemical Examination of Urine

Reagent Strips

• They 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.
• Reagent strips consist of chemical-impregnated absorbent pads attached to a plastic that produce a chemical reaction in contact with urine.
  • The reactions are interpreted by comparing the color produced on the pad with a chart supplied by the manufacturer containing a semiquantitative value of degree (e.g. trace, 1+, 2+, 3+, or 4+) or an estimate of the milligrams per deciliter.
  • Some are automated and report in IS units.
• The two major types of reagent strips are manufactured under the tradenames Multistix (Siemens Medical Solutions Diagnostics, Tarrytown, N.Y.) and Chemstrip (Roche Diagnostics, Indianapolis, Ind.).
  • These products are available with single or multiple-testing areas, and the brand and number of tests used are a matter of laboratory preference.

Reagent Strip Technique

• To use, the strip is dipped completely but briefly into a well-mixed specimen, with excess urine removed by running the edge of the strip on the container when withdrawing it from the specimen. After waiting to allow reactions to take place, it is compared against the manufacturer’s chart (but not on the chart itself) using a good light source.
  • Formed elements such as red and white blood cells sink to the bottom of the specimen and may be undetected in an unmixed specimen.
  • Excess urine or dipping in urine leaches off the reagent chemicals, distorting color results and contaminating the specimen.
  • Blotting the edge of the strip on absorbent paper and holding the strip horizontally while comparing it with the color chart is recommended.
  • Manufacturers usually provide timings for readings; however, when precise timing cannot be achieved, manufacturers recommend that reactions be read between 60 and 120 seconds.
  • Reagent strips and color charts from different manufacturers are not interchangeable.

Handling and Storage of Reagent Strips

• They are packaged in opaque containers with a desiccant at room temperature below 30°C to protect them from light, heat and moisture.
• Reagent strip bottles are removed just prior to testing and tightly resealed immediately. When opening, it should be away from the presence of volatile fumes.
• All bottles are stamped with an expiration date that represents the functional life expectancy of the chemical pads.
• Care must be taken not to touch the chemical pads when removing the strips.

Quality Control of Reagent Strips

• Reagent strips must be checked with both positive and negative controls a minimum of once every 24 hours, or if the strips are new, have questionable results and there are concerns on its integrity.
• Interfering substances in the urine, technical carelessness, and color blindness also produce errors.
• Tablets and liquid chemicals can be alternatives if questionable results are obtained or highly pigmented specimens are encountered, though they are less specific and sensitive.

pH

• A double-indicator system of methyl red and bromthymol blue is used to give a more specific measurement within the pH range of 5 to 9.
  • Methyl red produces a color change from red to yellow in the pH range 4 to 6, and bromthymol blue turns from yellow to blue in the range of 6 to 9.
• No known substances interfere with urinary pH measurements performed by reagent strips.

Protein

• It is based on the protein error of indicators where protein is able to alter the color of acid-base indicators without changing the pH.
  • Albumin is the main protein detected because it has more amino acid groups to accept hydrogen ions from the indicator.
• The strip contains either tetrabromphenol blue or 3′, 3′′, 5′, 5′′-tetrachlorophenol-3, 4, 5, 6-tetrabromosulfonphthalein, and an acid buffer to maintain the pH at a constant level.
  • If no protein is present, it appears yellow; however, as concentration increases, the color progresses through various shades of green and finally to blue.
  • Readings are reported in terms of negative, trace, 1+, 2+, 3+, and 4+; or the semiquantitative values of 30, 100, 300, or 2000 mg/dL corresponding to each color change.
  • Trace values are considered to be less than 30 mg/dL.
  • Interpretation of trace readings can be difficult and may be a laboratory option.
  • The specific gravity of the specimen should be considered because a trace protein in a dilute specimen is more significant than in a concentrated specimen.
• There are multiple semiquantitative reagent strip methods using immunochemical assays for albumin and creatinine.
  • Micral-Test reagent strips contain a gold-labeled antihuman albumin antibody-enzyme conjugate.
  • Albumin binds with the antibody and the resulting conjugates move up the strip to an enzyme substrate, causing a reaction which produces colors ranging from white to red equivalent to values from 0 to 10 mg/dL.
  • The Immunodip reagent strip uses an immunochromographic technique.
  • The strip is in a container which the urine is inserted into. It is coated with blue latex particles coated with antihuman albumin antibody which albumin binds onto.
  • As urine passes through the strip, substances that do not bind with the particles stop and form a blue band while albumin continues moving up and forms a second blue band.
    • A darker bottom band represents greater than 1.2 mg/dL, equal band colors represent 1.2 to 1.8 mg/dL, and a darker top band represents 2.0 to 8.0 mg/dL of albumin.
  • Clinitek Microalbumin reagent strips and Multistix Pro reagent strips provide simultaneous measurement of albumin/protein and creatinine that permits an estimation of the 24-hr microalbumin excretion.
  • By comparing the albumin excretion to the creatinine excretion, the albumin reading can be corrected for overhydration and dehydration in a random sample.
  • The albumin test pad is changed to a dye-binding reaction that is more specific for albumin than the protein error of indicators reaction.
  • Albumin reagent strips utilize the dye bis(3′,3′′, diodo-4′,4′′- dihydroxy-5′,5′′-dinitrophenyl)-3,4,5,6-tetra-bromosulphonphtalein (DIDNTB), which has a higher sensitivity and specificity for albumin.
  • It can measure albumin between 8 and 20 mg/dL (80 to 200 mg/L) without inclusion of other proteins.
    • Results are reported as 10 to 150 mg/L (1 to 15 mg/dL), with colors ranging from pale green to aqua blue.
  • Using paper treated with bis-(heptapropylene glycol) carbonate prevents interference from highly buffered alkaline urine.
  • The addition of polymethyl vinyl ether decreases the nonspecific binding of polyamino acids to the albumin pad.
  • Falsely elevated results can be caused by visibly bloody urine, and abnormally colored urines may interfere with the readings.
  • Reagent strips for creatinine contain copper sulfate (CuSO4, 3,3′,5,5′-tetramethylbenzidine (TMB) and diisopropyl benzene dihydroperoxide (DBDH).
  • The principle of the reagent strip for creatinine is based on the pseudoperoxidase activity of copper-creatinine complexes.
    • Creatinine in urine combines with the copper sulfate to form copper-creatinine peroxidase that then reacts with the peroxide DBDH, releasing oxygen ions that oxidize the chromogen TMB and producing a color change from orange through green to blue.
  • Results are reported as 10, 50, 100, 200, 300 mg/dL or 0.9, 4.4, 8.8, 17.7, or 26.5 mmol/L of creatinine.
    • No creatinine readings are considered abnormal, as creatinine is normally present in concentrations of 10 to 300 mg/dL (0.9 to 26.5 mmol/L).
    • Falsely elevated results can be caused by visibly bloody urine and the presence of the gastric acid–reducing medication cimetidine (Tagamet), and abnormally colored urines also may interfere with the readings.
  • The Bayer Multistix Pro 11 reagent strips include reagent pads for creatinine, protein-high and protein-low (albumin), along with pads for glucose, ketones, blood, nitrite, leukocyte esterase, pH, bilirubin, and specific gravity.
  • The protein-high reaction uses the protein error of indicators principle and the protein-low reaction is the previously discussed dye-binding method.
  • Results are reported as the protein:creatinine ratio, although the protein-low result is also included in the calculation.
    • Abnormal results for the A:C (albumin:creatinine) ratio are 30 to 300 mg/g or 3.4 to 33.9 mg/mmol.
• Possible sources of error include a highly buffered alkaline urine, contamination with quaternary ammonium compounds, detergents and antiseptics, a high specific gravity and a high amount of non-albumin proteins.
  • Highly buffered alkaline urine overrides the acid buffer system, producing a rise in pH and a color change unrelated to protein concentration.
  • It can be addressed by using the sulfosalicyclic acid (SSA) precipitation test or just acidifying the specimen itself.
  • Quaternary ammonium compounds and a high specific gravity cause a false-positive reading.

Glucose

• The two main reagent strips make use of glucose oxidase and copper reduction.
  • For glucose oxidase, the strip has peroxidase, chromogen and a buffer to produce a double sequential enzyme reaction.
  • Glucose oxidase catalyzes a reaction between glucose and room air to produce gluconic acid and peroxide which is then catalyzed by peroxidase to react with chromogen to form an oxidized colored compound that represents the presence of glucose.
    • Results are reported in terms of negative, trace, 1+, 2+, 3+, and 4+; however, the color charts also provide quantitative measurements ranging from 100 mg/dL to 2 g/dL, or 0.1% to 2%.
  • Reagent strip manufacturers use several different chromogens, including potassium iodide (green to brown) and tetramethylbenzidine (yellow to green).
  • Interference may occur from contamination with peroxide or strong oxidizing detergents, the presence of ascorbic acid and ketones, having a urine sample with a high specific gravity and low temperature, and improper storage.
    • To minimize interference from ascorbic acid, reagent strip manufacturers are incorporating additional chemicals into the test pads.
    • An example is iodate which can oxidize ascorbic acid.
    • High levels of ketones also affect glucose oxidase tests at low glucose concentrations, but they seldom present a problem.
    • High specific gravity and low temperature may decrease the sensitivity of the test.

Ketones

• Reagent strip tests use the sodium nitroprusside (nitroferricyanide) reaction to measure ketones.
  • In this reaction, acetoacetic acid in an alkaline medium reacts with sodium nitroprusside to produce a purple color.
  • It does not measure other ketones like beta-hydroxybutyric acid and acetone, but they don’t need to be tested and can be assumed to be present.
  • Results are reported qualitatively as negative, trace, small (1+), moderate (2+), or large (3+), or semiquantitatively as negative, trace (5 mg/dL), small (15 mg/dL), moderate (40 mg/dL), or large (80 to 160 mg/dL).

Blood

• Chemical tests for blood use the pseudoperoxidase activity of hemoglobin to catalyze a reaction between hydrogen peroxide and the chromogen tetramethylbenzidine to produce an oxidized chromogen, which has a green-blue color.
  • Reagent strip manufacturers incorporate peroxide, tetramethylbenzidine, and buffer into the blood testing area.
  • In the presence of free hemoglobin/myoglobin, uniform color ranging from a negative yellow through green to a strongly positive green-blue appear on the pad.
  • In contrast, intact red blood cells are lysed when they come in contact with the pad, and the liberated hemoglobin produces an isolated reaction that results in a speckled pattern on the pad.
• Reagent strip tests can detect concentrations as low as five red blood cells per microliter; however, care must be taken when comparing these figures with the actual microscopic values, because the absorbent nature of the pad attracts more than 1 mL of urine.
  • The terms trace, small, moderate, and large or trace, 1+, 2+ and 3+ are used for reporting.
• Possible interfering agents include menstrual contamination, strong oxidizing detergents, vegetable peroxidase and bacterial enzymes, ascorbic acid, high specific gravity, formalin or hypertension medication, and unmixed specimens.
  • Sediments containing bacteria should be checked closely for the presence of red blood cells.
  • Both Multistix and Chemstrip have modified their reagent strips to reduce this interference to very high levels (25 mg/dL) of ascorbic acid.
  • Multistix uses a peroxide that is less subject to reduction by ascorbic acid.
  • Chemstrip overlays the reagent pad with an iodate-impregnated mesh that oxidizes the ascorbic acid prior to its reaching the reaction pad.
  • A specimen with high specific gravity contains crenated red blood cells that do not lyse when they come in contact with the reagent pad.
  • Decreased reactivity may be seen when formalin is used as a preservative or when the hypertension medication, captopril, or high concentrations of nitrite (greater than 10 mg/dL) are present.
  • Red blood cells settle to the bottom of the specimen container, and failure to mix the specimen prior to testing causes a falsely decreased reading.

Bilirubin

• Routine testing for urinary bilirubin by reagent strip uses the diazo reaction.
  • Bilirubin combines with 2,4-dichloroaniline diazonium salt or 2,6-dichlorobenzene-diazonium-tetrafluoroborate in an acid medium to produce an azodye, with colors ranging from increasing degrees of tan or pink to violet, respectively.
  • Qualitative results are reported as negative, small, moderate, or large, or as negative, 1+, 2+, or 3+.
  • Reagent strip color reactions for bilirubin are more difficult to interpret than other reagent strip reactions and are easily influenced by other pigments present in the urine.
  • Atypical color reactions are frequently noted on visual examination and are measured by automated readers.

Urobilinogen

• Multistix uses Ehrlich’s aldehyde reaction, in which urobilinogen reacts with p-dimethylaminobenzaldehyde (Ehrlich reagent) to produce colors ranging from light to dark pink.
  • Results are reported as Ehrlich units (EU), which are equal to mg/dL, ranging from normal readings of 0.2 and 1 through abnormal readings of 2, 4, and 8.
  • Reaction interferences include Ehrlich reactive compounds, temperature, highly pigmented urine, improper preservation or preservation using formalin, and timing of urine collection after meals.
  • False-positive reactions may occur in the presence of porphobilinogen, indican, p-aminosalicylic acid, sulfonamides, methyldopa, procaine, and chlorpromazine compounds.
    • The presence of porphobilinogen is clinically significant; however, the reagent strip test is not considered a reliable method to screen for its presence.
  • The sensitivity of the Ehrlich reaction increases with temperature, and testing should be performed at room temperature.
  • As a result of increased excretion of bile salts, urobilinogen results are normally highest following a heavy meal.
  • False-negative results occur most frequently when specimens are improperly preserved, allowing urobilinogen to be photo-oxidized to urobilin.
• Chemstrip incorporates an azo-coupling (diazo) reaction using 4-methoxybenzene-diazonium-tetrafluoroborate to react with urobilinogen, producing colors ranging from white to pink.
  • This reaction is more specific for urobilinogen than the Ehrlich reaction. Results are reported in mg/dL.
  • Reaction interferences include highly pigmented urine, improper preservation or preservation using formalin, high concentrations of nitrite and timing of urine collection after meals.
  • High concentrations of nitrite interfere with the azo-coupling reaction on Chemstrip.

Nitrite

• Nitrite is detected by the Greiss reaction, in which nitrite at an acidic pH reacts with an aromatic amine (para-arsanilic acid or sulfanilamide) to form a diazonium compound that then reacts with tetrahydrobenzoquinolin compounds to produce a pinkcolored azodye.
  • The chemical basis of the nitrite test is the ability of certain bacteria to reduce nitrate, a normal constituent of urine, to nitrite, which does not normally appear in the urine.
  • Although different shades of pink may be produced, the test does not measure the degree of bacteriuria, and any shade of pink is considered to represent a clinically significant amount of bacteria.
  • Results are reported only as negative or positive.
• The sensitivity of the test is standardized to correspond with a quantitative bacterial culture criterion of 100,000 organisms per milliliter.
• However, it has limitations such as only detecting gram-negative bacteria, only being suitable for first morning specimens or specimens collected after urine has remained in the bladder for at least 4 hours, and a dependency on the patient’s diet.
  • Bacteria that lack the enzyme reductase, such as gram-positive bacteria and yeast, do not possess the ability to reduce nitrate to nitrite.
  • Bacteria capable of reducing nitrate must remain in contact with the urinary nitrate long enough to produce nitrite.
  • There is a possibility of a false-negative result owing to lack of dietary nitrate from produce and processed meats.
  • Further reduction of nitrite to nitrogen may occur when large numbers of bacteria are present.
• Reaction interferences include inhibition of bacterial metabolism by the presence of antibiotics, large quantities of ascorbic acid, decreased sensitivity in specimens with a high specific gravity, improper storage of specimens and highly pigmented specimens.
  • Ascorbic acid interferes with the diazo reaction as it competes with nitrite to combine with the diazonium salt.
  • Contaminant bacteria produces measurable amounts of nitrite on specimens left to stand unpreserved, giving a false positive result.
  • Highly pigmented urines produce atypical color reactions.

Leukocyte Esterase

• The reagent strip reaction uses the action of LE to catalyze the hydrolysis of an acid ester embedded on the reagent pad to produce an aromatic compound and acid, which then combine with a diazonium salt present on the pad to produce a purple azodye.
  • The LE reaction requires the longest time of all the reagent strip reactions (2 minutes).
  • Reactions are reported as trace, small, moderate, and large or trace, 1+, 2+ and 3+.
  • Trace readings may not be significant and should be repeated on a fresh specimen.
• Reaction interferences are caused by strong oxidizing agents or formalin, highly pigmented urines and the presence of nitrofurantoin, high concentrations of protein (greater than 500 mg/dL), glucose (greater than 3 g/dL), oxalic acid and ascorbic acid, high specific gravity and the presence of antibiotics.
  • The presence of strong oxidizing agents or formalin in the collection container causes false-positive reactions.
  • Highly pigmented urines and the presence of nitrofurantoin obscure the color reaction.
  • False-negative results may occur in the presence of high concentrations of protein, glucose, ascorbic acid and oxalic acid.
  • Ascorbic acid also combines with the diazonium salt.
  • Crenation of leukocytes preventing release of esterases may occur in urines with a high specific gravity.
  • The presence of the antibiotics gentamicin, cephalexin, cephalothin, and tetracycline decreases the sensitivity of the reaction.

Specific Gravity

• The reagent strip reaction is based on the change in pKa (dissociation constant) of a polyelectrolyte in an alkaline medium.
  • The polyelectrolyte ionizes, releasing hydrogen ions in proportion to the number of ions in the solution.
  • The higher the concentration of urine, the more hydrogen ions are released, thereby lowering the pH. Incorporation of the indicator bromthymol blue on the reagent pad measures the change in pH.
  • As the specific gravity increases, the indicator changes from blue (1.000 [alkaline]), through shades of green, to yellow (1.030 [acid]).
  • Readings can be made in 0.005 intervals by careful comparison with the color chart.
• Reaction interferences include increased protein and pH.
  • Elevated concentrations of protein slightly increase the readings as a result of protein anions.
  • Specimens with a pH of 6.5 or higher have decreased readings caused by interference with the bromthymol blue indicator (the blue-green readings associated with an alkaline pH correspond to a low specific gravity reading).
  • Manufacturers recommend adding 0.005 to specific gravity.

pH

• A healthy individual usually produces a first morning specimen with a slightly acidic pH of 5.0 to 6.0, but it increases following meals (alkaline tide).

  • However, normal random samples have no standard pH and can range from 4.5 to 8.0, and it must be considered in conjunction with other patient information, such as the acid-base content of the blood, the patient’s renal function, the presence of a urinary tract infection, the patient’s dietary intake, and the age of the specimen.
  • The pH of freshly excreted urine does not reach 9 in normal or abnormal condition and indicates an improperly preserved specimen.

  Clinical Significance

  • It can aid in determining the existence of systemic acid-base disorders of metabolic or respiratory origin and in the management of urinary conditions that require the urine to be maintained at a specific pH.
  • In respiratory or metabolic acidosis or alkalinosis not related to renal function disorders, the urine follows the pH of the body, so this knowledge can differentiate the condition or organ affected.
  • The precipitation of crystals and minerals depend on the pH, aiding in microscopic examination.
  • The maintenance of acidic urine may be useful in controlling UTIs because their multiplication is hampered in such environment.
  • Persons on high-protein and high-meat diets tend to produce acidic urine, whereas urine from vegetarians is more alkaline, owing to the formation of bicarbonate following digestion of many fruits and vegetables.
  • Cranberry juice, which produces an acidic urine, has traditionally been used as a home remedy for minor bladder infections.
  • Medications, like methenamine mandelate (Mandelamine) and fosfomycin tromethamine, metabolize to acidify urine.

Protein

• Normal urine contains very little protein and less than 10 mg/dL or 100 mg per 24 hours is excreted.
  • They comprise of proteins with low molecular weights and proteins produced in the genitourinary tract.
  • Specifically, they are albumin, serum and tubular microglobulins, Tamm-Horsfall proteins produced by the tubules, and proteins from prostatic, seminal, and vaginal secretions.

Clinical Significance

• The presence of proteinuria is often associated with early renal disease, making the urinary protein test an important part of any physical examination.
  • Clinical proteinuria is indicated at ≥30 mg/dL (300 mg/L).
  • It does not always signify renal disease; however, its presence does require additional testing to determine whether the protein represents a normal or a pathologic condition.
• The causes of proteinuria are varied and can be grouped into three major categories: pre-renal, renal, and post-renal, based on the origin of the protein.
  • Pre-renal proteinuria is caused by conditions affecting the plasma prior to its reaching the kidney and, therefore, is not indicative of actual renal disease.
  • This is caused by increased levels of plasma proteins with low molecular weights such as hemoglobin, myoglobin and acute phase reactants associated with infection and inflammation that all exceed the normal reabsorptive capacity of the renal tubules.
    • The amount of protein that appears in the urine following glomerular damage ranges from slightly above normal to 4 g/day.
    • An example is in people with multiple myeloma, a proliferative disorder of the immunoglobulin-producing plasma cells where the serum contains markedly elevated levels of monoclonal immunoglobulin light chains or Bence-Jones protein.
    • Bence-Jones protein coagulates at temperatures between 40°C and 60°C and dissolves when the temperature reaches 100°C, so it makes the specimen turbid before becoming clear
      • Interference due to other proteins can be removed by filtering the specimen at 100°C and observing the specimen for turbidity as it cools to between 40°C and 60°C.
    • However, not all persons with multiple myeloma excrete detectable levels of Bence Jones protein. The main diagnosis is through serum electrophoresis and immunoelectrophoresis.
  • It is not detected in routine analysis with reagent strips.
  • Renal protenuria is the result of either glomerular or tubular damage, orthostatic/postural causes, or diabetic neuropathy.
  • If the glomerolus is damaged, filtration is impaired and increased amounts of serum protein, RBCs and WBCs pass trough the urine.
    • Possible causes include abnormal substances and increased blood pressure.
    • Amyloid material, toxic substances, and immune complexes found in lupus erythematosus and streptococcal glomerulonephritis damage the glomerolus.
    • Exercise, dehydration, hypertension and pre-eclampsia can increase blood pressure.
  • If the tubules are damaged, proteins of low molecular weights are not returned to the bloodstream.
    • Possible causes include exposure to toxic substances and heavy metals, severe viral infections and Fanconi syndrome, an inherited tubular defect
    • Increased albumin is usually present, but markedly elevated protein levels are seldom seen in tubular disorders.
  • Orthostatic proteinuria is caused by increased pressure on the renal vein that overrides the filtration capacity of the glomerolus from assuming a vertical position.
    • Patients suspected of orthostatic proteinuria are requested to empty their bladder before going to bed, collect a specimen immediately upon arising in the morning, and collect a second specimen after remaining in a vertical position for several hours.
    • If orthostatic proteinuria is present, a negative reading will be seen on the first morning specimen, and a positive result will be found on the second specimen.
  • Diabetic nephropathy impairs filtration and eventually causes failure due to increased glucose that go beyond the renal threshold.
    • It can be detected by the presence of microalbuminuria or low levels of albumin in the urine.
    • Albumin is produced in the liver, and impaired production may also indicate impaired production of insulin.
    • It is tested using 24-hour specimens reported in mg of albumin/24 hours or as the albumin excretion (AER) in μg/min, and it is is considered significant when 30 to 300 mg of albumin is excreted in 24 hours or the AER is 20-200 μg/min.
  • Post-renal proteinuria is caused when urine passes through the structures of the lower urinary tract (ureters, bladder, urethra, prostate, and vagina).
  • Bacterial and fungal infections and inflammations produce exudates containing protein from the interstitial fluid.
  • The presence of blood as the result of injury or menstrual contamination contributes protein, as does the presence of prostatic fluid and large amounts of spermatozoa.

Sulfosalicylic Acid (SSA) Precipitation Test

• Compared to reagent strips, it reacts to all forms of proteins; however, it must be performed on centrifuged specimens to remove any extraneous contamination as it may react to non-protein substances.
  • Radiographic dye increases the specific gravity and turbidity due to the precipitation of crystals.
  • Tolbutamide and antibiotics like cephalosporins, penicillins and sulfonamides can also affect the test.
• To address highly buffered alkaline urine, a more concentrated solution of SSA may be used.

Glucose

Clinical Significance

• The blood level at which tubular reabsorption stops (renal threshold) for glucose is approximately 160 to 180 mg/dL; however, it doesn’t always indicate diabetes.
  • A normal person may have glycosuria or glucose in the urine following a meal with a high glucose content.
  • During pregnancy, hormones secreted by the placenta block the action of insulin, resulting in insulin resistance and hyperglycemia. It eventually disappears after delivery.
  • However, glucose can cross the placenta and cause the baby’s pancreas to produce more insulin, eventually putting the cells under insulin resistance and storing glucose as fat. This makes the baby larger and puts it at a higher risk of obesity and type 2 diabetes.
  • Women who have gestational diabetes also are prone to developing type 2 diabetes mellitus in later years.
  • Hormonal disorders such as pancreatitis, pancreatic cancer, acromegaly, Cushing syndrome, hyperthyroidism and pheochromocytoma can also cause glycosuria.
  • The hormones glucagon, epinephrine, cortisol, thyroxine and growth hormone work in opposition to insulin and cause the breakdown of glycogen to glucose (glycogenolysis), resulting in increased levels of circulating glucose.
  • Cerebrovascular trauma and myocardial infarction subjects the body to severe stress and increases epinephrine levels, causing glycosuria.

Copper Reduction/Benedict’s Test

• The test relies on the ability of glucose and other substances to reduce copper sulfate to cuprous oxide in the presence of alkali and heat.
  • A color change progressing from a negative blue (CuSO4) through green, yellow, and orange/red (Cu2O) occurs when the reaction takes place
• A more convenient method that employs Benedict’s principle is the Clinitest tablet.
  • The tablets contain copper sulfate, sodium carbonate, sodium citrate, and sodium hydroxide.
  • Upon addition of the tablet to water and urine, heat is produced by the hydrolysis of sodium hydroxide and its reaction with sodium citrate, and carbon dioxide is released from the sodium carbonate to prevent room air from interfering with the reduction reaction.
    • Tubes should be placed in a rack and not held in the hand because the reaction heat could cause a burn.
    • Care must be taken to observe the reaction closely as it is taking place, because at high glucose levels, a phenomenon known as “pass through” may occur.
    • The color produced passes through the orange/red stage and returns to a green-brown color, and if not observed, a high glucose level may be reported as negative.
    • An alternate method using two drops instead of five drops of urine can minimize the occurrence of “pass through”, but a separate color chart must be used to interpret the reaction.
    • At the conclusion of the effervescent reaction, the tube is gently shaken, and the color ranging from blue to orange/red can be compared with the manufacturer’s color chart to determine the approximate amount of reducing substance.
  • The sensitivity of Clinitest to glucose is reduced to a minimum of 200 mg/dL, and it is subject to interference from other reducing sugars, including galactose, lactose, fructose, maltose, pentoses, ascorbic acid, certain drug metabolites, and antibiotics such as cephalosporins.
    • If a negative glucose oxidase strip and positive Clinitest results are found, galactosuria may be present.
    • Galactose in the urine of a newborn represents an inborn error of metabolism in which lack of the enzyme galactose-1-phosphate uridyl transferase prevents breakdown of ingested galactose and results in failure to thrive and other complications, including death.
  • Clinitest tablets are very hygroscopic and should be stored in their tightly closed packages.
    • A strong blue color in the unused tablets and vigorous tablet fizzing suggests deterioration due to moisture accumulation.

Ketones

• They represent the three intermediate products of fat metabolism: acetone, acetoacetic acid and betahydroxybutyric acid.
  • The three ketone compounds are not present in equal amounts in urine.
  • The proportions of 78% beta-hydroxybutyric acid, 20% acetoacetic acid and 2% acetone are relatively constant in all specimens.
  • Both acetone and beta-hydroxybutyric acid are produced from acetoacetic acid

Clinical Significance

• They don’t usually appear in urine or are in insignificant levels as all metabolized fat is completely broken down into carbon dioxide and water; however, when carbohydrates are unmetaboliozed, removed or inadequate, stored fat is used and then ketones will be detected.
  • Testing for urinary ketones is most valuable in the management and monitoring of insulin-dependent (type 1) diabetes mellitus.
  • As glucose cannot be metabolized, the body is forced to use fat.
  • Increased accumulation of ketones in the blood leads to electrolyte imbalance, dehydration, and, if not corrected, acidosis and eventual diabetic coma.
  • To aid in the monitoring of diabetes, ketone tests are not only included in all multiple-test strips, but are also combined with glucose on strips used primarily for at-home testing by patients diagnosed with diabetes.
  • Non-diabetic ketonuria is mostly caused by inadequate intake or an accelerated loss of food (e.g. vomiting) in patient’s with illnesses.
  • Frequent strenuous exercise can cause overuse of available carbohydrates.

Acetest Tablets

• In cases of severe ketosis, it may be necessary to perform tests on serial dilutions to provide more information as to the extent of ketosis.
• It provides sodium nitroprusside, glycine, disodium phosphate, and lactose in tablet form.
  • The addition of lactose gives better color differentiation.
• They can also be used for serum and other body fluid testing.
• Acetest tablets are hygroscopic, so if the specimen is not completely absorbed within 30 seconds, a new tablet should be used.

Reaction Interference

• Specimens obtained following diagnostic procedures using the dyes phenolsulfonphthalein and bromsulphalein may produce an interfering red color in the alkaline test medium, as does highly pigmented red urine.
• Large amounts of levodopa and medications containing sulfhydryl groups, including mercaptoethane sulfonate sodium (MESNA) and captopril, may produce atypical color reactions.
• Reactions with interfering substances frequently fade on standing, whereas color development from acetoacetic acid increases, resulting in false-positive results from improperly timed readings.
• Falsely decreased values due to the volatilization of acetone and the breakdown of acetoacetic acid by bacteria are seen in improperly preserved specimens.

Bilirubin

Clinical Significance

• The appearance of bilirubin in the urine can provide an early indication of liver disease.
  • It is often detected long before the development of jaundice.
  • This determination can be even more significant with blood bilirubin.
  • Jaundice due to increased destruction of red blood cells does not produce bilirubinuria. This is because the serum bilirubin is present in the unconjugated form and the kidneys cannot excrete it.
• Conjugated bilirubin appears in the urine when the normal degradation cycle is disrupted by obstruction of the bile duct (e.g., gallstones or cancer) or when the integrity of the liver is damaged, allowing leakage of conjugated bilirubin into the circulation.

Ictotest Tablets

• The Ictotest is less subject to interference and is sensitive to 0.05 to 0.10 mg/dL of bilirubin.
• An Ictotest may be requested to detect early stages of liver disease.
• Ictotest kits consist of testing mats and tablets containing p-nitrobenzene-diazonium-ptoluenesulfonate, SSA, sodium carbonate, and boric acid.
  • Ten drops of urine are added to the mat, which has special properties that cause bilirubin to remain on the surface as the urine is absorbed.
  • Following the chemical reaction, a blue-to-purple color appears on the mat when bilirubin is present.
  • Colors other than blue or purple appearing on the mat are considered to be a negative result.
  • If interference in the Ictotest is suspected, it can usually be removed by adding water directly to the mat after the urine has been added.
  • Interfering substances are washed into the mat, and only bilirubin remains on the surface.

Reaction Interference

• A particular concern are the yelloworange urines from persons taking phenazopyridine compounds, because the thick pigment produced may be mistaken for bilirubin on initial examination.
• The presence of indican and metabolites of the medication Lodine may cause false-positive readings.
• The false-negative results caused by the testing of specimens that are not fresh are the most frequent errors associated with bilirubin testing.
  • Bilirubin is an unstable compound that is rapidly photo-oxidized to biliverdin when exposed to light. Biliverdin does not react with diazo tests.
• False-negative results also occur when hydrolysis of bilirubin diglucuronide produces free bilirubin, because this is less reactive in the reagent strip tests.
• High concentrations of ascorbic acid (greater than 25 mg/dL) and nitrite may lower the sensitivity of the test, because they combine with the diazonium salt and prevent its reaction with bilirubin.

Urobilinogen

Clinical Significance

• Increased urine urobilinogen (greater than 1 mg/dL) is seen in liver disease and hemolytic disorders
  • An increased amount unconjugated bilirubin the liver processes also produces an increased amount of urobilinogen for the liver to process as well, overworking it and keeping the urobilinogen in the blood until it is filtered by the kidneys.
  • The absence of urobilinogen in the urine and feces is also diagnostically significant and represents an obstruction of the bile duct that prevents the normal passage of bilirubin into the intestine.
  • An additional observation is the production of pale stools as the result of the lack of urobilin.

Ehrlich Tube Test

• In the tube method, one part Ehrlich reagent was added to 10 parts of urine, along with sodium acetate to enhance the color reaction.
  • A sample positive for urobilinogen is expected to have a cherry-red color.
  • However, it is subject to false-positive results in the presence of porphobilinogen and Elrich reactive compounds, so the Watson-Schwartz Differentiation Test is used.
  • In this test, two tubes contain both 2 mL of urine and 4 mL of sodium acetate, but one has 2 mL chloroform and the other has 2 mL butanol.
    • The addition of chloroform to Tube 1 results in the extraction of urobilinogen into the chloroform (bottom) layer, producing a colorless urine (top) layer, and a red chloroform layer on the bottom.
    • Neither porphobilinogen nor other Ehrlich-reactive compounds are soluble in chloroform.
    • The addition of butanol to Tube 2 produces a red (upper) butanol layer if urobilinogen or Ehrlich-reactive compounds are present and a colorless butanol layer if porphobilinogen is present.
    • Porphobilinogen is also not soluble in butanol; however, urobilinogen and other Ehrlich-reactive compounds are extracted into butanol.
    • If both urobilinogen and porphobilinogen are present, both layers appear red.
      • Before reporting the test as positive for both substances, an additional chloroform extraction should be performed on the red urine (upper) layer in Tube 1 to ensure that the red color is not due to excess urobilinogen.

Nitrite

Clinical Significance

• The nitrite test is valuable for detecting initial bladder infection (cystitis), because patients are often asymptomatic or have vague symptoms that would not lead the physician to order a urine culture.
  • Pyelonephritis, an inflammatory process of the kidney and adjacent renal pelvis, is a frequent complication of untreated cystitis and can lead to renal tissue damage, impairment of renal function, hypertension, and even septicemia.
• The nitrite test also can be used to evaluate the success of antibiotic therapy and to periodically screen persons with recurrent infections, patients with diabetes, and pregnant women, all of whom are considered to be at high risk for UTI.
• Many laboratories use the nitrite test in combination with the leukocyte esterase test to determine the necessity of performing urine cultures.

Leukocyte Esterase (LE)

Clinical Significance

• Normal values for leukocytes are based on the microscopic sediment examination and vary from 0 to 2 to 0 to 5 per high power field.
  • Women tend to have higher numbers than men as a result of vaginal contamination.
  • Increased urinary leukocytes are indicators of UTI.
• Tests detect the presence of esterase in granulocytic white blood cells (neutrophils, eosinophils, and basophils) and monocytes, and in histocytes and Trichomonas protists.
  • Neutrophils are the leukocytes most frequently associated with bacterial infections.
  • Lymphocytes, erythrocytes, bacteria, and renal tissue cells do not contain esterases.
  • However, the disease associated with a positive result may be accompanied by bacteria.
    • Infections caused by Trichomonas, Chlamydia, yeast, and inflammation of renal tissues (i.e., interstitial nephritis) produce leukocyturia without bacteriuria.