Chemical Exam Of Urine PDF

 CHAPTER 6 Chemical Examination of Urine LEARNING OUTCOMES Upon completing this chapter, the reader will be able to: 6-1 Describe the proper technique for performing reagent 6-1...

 CHAPTER 6 Chemical Examination of Urine LEARNING OUTCOMES Upon completing this chapter, the reader will be able to: 6-1 Describe the proper technique for performing reagent 6-14 Differentiate among hematuria, hemoglobinuria, and strip testing. myoglobinuria with regard to the appearance of urine and serum, as well as the clinical significance of each. 6-2 List four causes of premature deterioration of reagent strips, and describe how to avoid them. 6-15 Describe the chemical principle of the reagent strip method for blood testing, and list possible causes of 6-3 List five quality-control procedures routinely per- interference. formed with reagent strip testing. 6-16 Outline the steps in the degradation of hemoglobin to 6-4 List the reasons for measuring urinary pH, and discuss bilirubin, urobilinogen, and urobilin. their clinical applications. 6-17 Describe the relationship of urinary bilirubin and uro- 6-5 Discuss the principle of pH testing by reagent strip. bilinogen to each of the following diagnoses: bile duct 6-6 Differentiate among prerenal, renal, and postrenal pro- obstruction, liver disease, and hemolytic disorders. teinuria, and give clinical examples of each. 6-18 Discuss the principle of the reagent strip test for uri- 6-7 Explain the “protein error of indicators,” and list any nary bilirubin, including possible sources of error. sources of interference that may occur with this 6-19 State two reasons for increased urine urobilinogen and method of protein testing. one reason for a decreased urine urobilinogen. 6-8 Discuss microalbuminuria, including significance, 6-20 Discuss the principle of the nitrite reagent strip test for reagent strip tests, and their principles. bacteriuria. 6-9 Explain why glucose that is normally reabsorbed in the 6-21 List five possible causes of a false-negative result proximal convoluted tubule may appear in the urine, in the reagent strip test for nitrite. and state the renal threshold levels for glucose. 6-22 State the principle of the reagent strip test for 6-10 Describe the principle of the glucose oxidase method leukocytes. of reagent strip testing for glucose, and name possible causes of interference with this method. 6-23 Discuss the advantages and sources of error of the reagent strip test for leukocytes. 6-11 Describe the copper reduction method for the detec- tion of urinary-reducing substances, and discuss the 6-24 Explain the principle of the chemical test for specific current use of this procedure. gravity. 6-12 Name the three “ketone bodies” appearing in urine 6-25 Compare reagent strip testing for urine specific gravity and three causes of ketonuria. with osmolality and refractometer testing. 6-13 Discuss the principle of the sodium nitroprusside reac- 6-26 Correlate physical and chemical urinalysis results. tion to detect ketones, including sensitivity and possi- ble causes of interference. 140 Part Two | Urinalysis KEY TERMS Ascorbic acid Greiss reaction Orthostatic proteinuria Bacteriuria Hematuria Postrenal proteinuria Bilirubin Hemoglobinuria Prerenal proteinuria Diazo reaction Hemosiderin Protein error of indicators Ehrlich reaction Jaundice Proteinuria Fanconi syndrome Ketonuria Renal proteinuria Ferritin Leukocyturia Stercobilinogen Glucosuria Microalbuminuria Urobilinogen Glycosuria Myoglobinuria Uromodulin Introduction Routine chemical examination of urine has changed dramati- cally since the early days of urine testing due to the develop- ment of the reagent strip method for chemical analysis. Commercial reagent strips currently provide a simple, rapid means for performing medically significant chemical analysis of urine, including pH, protein, glucose, ketones, blood, bilirubin, urobilinogen, nitrite, leukocytes, and specific grav- ity. The two major types of reagent strips are manufactured under the trade names Multistix (Siemens Healthcare Diagnos- tics, Inc., Tarrytown, NY) and Chemstrip (Roche Diagnostics, Indianapolis, IN). These products are available with single- or multiple-testing areas, and the brand and number of tests used Figure 6–1 Chemical examination of urine comparing reagent are a matter of laboratory preference. Certain variations relating strip color reactions using reagent strips consisting of chemical- to chemical reactions, sensitivity, specificity, and interfering impregnated absorbent pads attached to a plastic strip. (From substances occur among the products and are discussed in the Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, following sections. Reagent strip brands are also specified by 4th ed. FA Davis, Philadelphia, 2019.) instrumentation manufacturers. the edge of the strip on the container when withdrawing Reagent Strips it from the specimen, blotting the strip horizontally on an ab- sorbent medium, waiting the specified length of time for reactions Chemical reagent strips consist of chemical-impregnated ab- to take place according to the manufacturer, and then comparing sorbent pads attached to a plastic strip (Fig. 6-1). A color- the colored reactions against the manufacturer’s chart using a producing chemical reaction takes place when the absorbent good light source. pad comes in contact with urine. The reactions are interpreted Errors Caused by Improper Technique by comparing the color produced on the pad within the required time frame with a chart supplied by the manufacturer (Fig. 6-2). 1. Formed elements, such as red and white blood cells, Several colors or intensities of a color for each substance being sink to the bottom of the specimen and will be unde- tested appear on the chart. By careful comparison of the colors tected in an unmixed specimen. on the chart with the strip, a semiquantitative value of trace, 2. Allowing the strip to remain in the urine for an 1+, 2+, 3+, or 4+ can be reported. An estimate of the mil- extended period may cause leaching of reagents ligrams per deciliter present is available for appropriate testing from the pads. areas. Automated reagent strip readers also provide Système 3. Allowing excess urine to remain on the strip after its International (SI) units. removal from the specimen can produce a runover between chemicals on adjacent pads, producing distor- Reagent Strip Technique tion of the colors. To ensure against runover, blotting the The testing methodology includes dipping the reagent strip edge of the strip on absorbent paper and holding the completely, but briefly, into a well-mixed specimen at room strip horizontally while comparing it with the color temperature, removing excess urine from the strip by running chart is recommended.  Chapter 6 | Chemical Examination of Urine 141 5. A good light source is essential for accurate interpreta- tion of color reactions. 6. The strip must be held close to the color chart without actually being placed on the chart. Manufacturers vary in the direction of the reagent strip to the color chart on the container when reading results. Automated reagent strip instruments standardize the color interpretation and timing of the reaction and are not subject to room lighting deficiencies or inconsistency among laboratory personnel (Chapter 2). 7. Reagent strips and color charts from different manufac- A turers are not interchangeable. 8. Specimens that have been refrigerated must be allowed to return to room temperature before reagent strip testing, as the enzymatic reactions on the strips are temperature dependent. Handling and Storing Reagent Strips In addition to using the correct testing technique, reagent strips must be protected from deterioration caused by moisture, volatile chemicals, heat, and light. Reagent strips are packaged in opaque containers with a desiccant to protect them from light and moisture. Strips are removed just before testing, and the bottle is tightly resealed immediately. Bottles should not be opened in the presence of volatile fumes. Manufacturers rec- ommend that reagent strips be stored at room temperature below 30°C (but never refrigerated). All bottles are stamped with an expiration date that represents the functional life ex- pectancy of the chemical pads. Reagent strips must not be used past the expiration date. Care must be taken not to touch the chemical pads when removing the strips. A visual inspection of the strip should be done each time a strip is used to detect deterioration, even though the strips may still be within the expiration date. PROCEDURE 6-1 Reagent Strip Technique1,2 Visit www.fadavis.com for Video 6-1 (Chemical testing of urine). B Procedure Figure 6–2 (A) Siemens Multistix 10 SG Reagent Strips Color Chart for Urinalysis. (B) Roche Chemstrip 10SG Reagent Strips Color Chart. 1. Dip the reagent strip briefly into a well-mixed, (Courtesy of Roche Diagnostics Corporation.) uncentrifuged urine specimen at room temperature. 2. Remove excess urine by touching the edge of the strip to the container as the strip is withdrawn. 3. Blot the edge of the strip on a disposable absorbent pad. 4. The timing for reactions to take place varies between tests and manufacturers and ranges from 30 seconds to 4. Wait the specified amount of time for the reaction to 120 seconds for leukocyte esterase (LE). For the best occur. semiquantitative results, the manufacturer’s stated time 5. Compare the color reaction of the strip pads to the should be followed; however, when precise timing can- manufacturer’s color chart in good lighting. not be achieved, manufacturers recommend that reac- 6. Read the results at the correct time, and record the tions be read between 60 and 120 seconds, with the LE results. reaction read at 120 seconds. 142 Part Two | Urinalysis Quality Control of Reagent Strips SUMMARY 61 Reagent Strip Testing Reagent strips must be checked with both positive and negative Care of Reagent Strips controls according to the frequency established by the labora- tory policy. Many laboratories perform this check at the begin- 1. Store with desiccant in an opaque, tightly closed ning of each shift. Testing is also performed when a new bottle container. of reagent strips is opened, questionable results are obtained, 2. Store below 30°C; do not freeze. or there is concern about the integrity of the strips. All quality 3. Do not expose to volatile fumes. control results must be recorded following laboratory protocol. 4. Do not use past the expiration date. Several companies manufacture both positive and negative controls. Distilled water is not recommended as a negative con- 5. Do not use if chemical pads become discolored. trol because reagent strip chemical reactions are designed to 6. Remove strips immediately before use. perform at ionic concentrations similar to urine. All readings of Technique the negative control must be negative, and positive control read- ings should agree with the published value. Results that do not 1. Mix specimen well. agree with the published values must be resolved through the 2. Let refrigerated specimens warm to room tempera- testing of additional strips and controls (see Chapter 1). ture before testing. Demonstration of chemically acceptable reagent strips 3. Dip the strip completely, but briefly, into the does not entirely rule out the possibility of inaccurate results. specimen. Interfering substances in the urine, technical carelessness, and 4. Remove excess urine by withdrawing the strip against color blindness also produce errors. Reagent strip manufactur- the rim of the container and by blotting the edge of ers have published information concerning the limitations the strip. (e.g., interfering substances, sensitivities) of their chemical reactions, and laboratory personnel should be aware of these 5. Compare reaction colors with the manufacturer’s conditions. As mentioned in Chapter 5, a primary example of chart under a good light source at the specified time. reagent strip interference is the masking of color reactions by 6. Perform confirmatory tests when indicated. the orange pigment present in the urine of people taking 7. Be alert for the presence of interfering substances. phenazopyridine compounds. If laboratory personnel do not 8. Understand the principles and significance of the test; recognize the presence of this pigment or other pigments, they read package inserts. will report many erroneous results. 9. Relate chemical findings to each other and to the Confirmatory Testing physical and microscopic urinalysis results. Confirmatory tests are procedures using different reagents or Quality Control methodologies to detect the same substances as detected by the 1. Test open bottles of reagent strips with known posi- reagent strips with the same or greater sensitivity or specificity.3 tive and negative controls per facility protocol. Nonreagent strip testing procedures using tablets and liquid 2. Resolve control results that are out of range by further chemicals may be available when questionable results are ob- testing. tained or highly pigmented specimens are encountered. In the past, many of these procedures were used routinely to confirm 3. Test reagents used in confirmatory tests with positive positive results. Increased specificity and sensitivity of reagent and negative controls. strips and the use of automated strip readers have reduced the 4. Perform positive and negative controls on new need for routine use of these procedures.3 The chemical reliabil- reagents and newly opened bottles of reagent strips. ity of these procedures also must be checked using positive and 5. Record all control results and reagent lot numbers. negative controls. Specific confirmatory tests are discussed in this chapter under their specific sections or the Historical Notes devoted to the chemical parameters for which they are used. The princi- phosphate, and weak organic acids and by the reabsorption ples and procedures for these tests are included to provide ad- of bicarbonate from the filtrate in the convoluted tubules (see ditional information on the principles of the reagent strips and Chapter 3). A healthy individual usually produces a first to provide the methodology to perform these tests if necessary. morning specimen with a slightly acidic pH of 5.0 to 6.0; a Facility protocol will determine the situations when they more alkaline pH is found after meals (alkaline tide). The should be performed. pH of normal random specimens can range from 4.5 to 8.0. Consequently, no normal values are assigned to urinary pH, pH and it must be considered in conjunction with other patient information, such as the acid–base content of the blood, the Along with the lungs, the kidneys are the major regulators of patient’s renal function, the presence of a urinary tract infec- the acid–base content in the body. They do this through the tion (UTI), the patient’s dietary intake, and the age of the secretion of hydrogen in the form of ammonium ions, hydrogen specimen (Table 6-1).  Chapter 6 | Chemical Examination of Urine 143 Table 6–1 Causes of Acid and Alkaline Urine formation of bicarbonate after digestion of many fruits and veg- etables. An exception to the rule is in patients who consume Acid Urine Alkaline Urine cranberry juice or supplements, which produce an acidic urine and have long been used as a home remedy for minor bladder Emphysema Hyperventilation infections because they inhibit the colonization of certain urinary Diabetes mellitus Vomiting pathogens. People who are prone to frequent UTIs are often ad- Starvation Renal tubular acidosis vised to drink cranberry juice or take over-the-counter cranberry Dehydration Presence of urease- pills. Medications prescribed for UTIs, such as methenamine producing bacteria mandelate (Mandelamine) and fosfomycin tromethamine (Monurol), are metabolized to produce an acidic urine. Diarrhea Vegetarian diet The pH of freshly excreted urine does not reach above 8.5 Presence of acid-producing Old specimens in normal or abnormal conditions. A pH above 8.5 is associ- bacteria (Escherichia coli) ated with a specimen that has been preserved improperly and High-protein diet indicates that a fresh specimen should be obtained to ensure Cranberry juice the validity of the analysis. Medications (methenamine mandelate Technical Tip 6-1. Collecting specimens in containers [Mandelamine], other than the single-use laboratory-supplied contain- fosfomycin ers can produce a pH above 8.5 if alkaline detergent tromethamine remains in the container. [Monurol]) Reagent Strip Reactions Clinical Significance The Multistix and Chemstrip brands of reagent strips measure urine pH in 0.5- or 1-unit increments between pH 5 to 8.5 visu- The importance of urinary pH is primarily as an aid in deter- ally and pH 5 to 9 instrumentally.2 To differentiate pH units mining the existence of systemic acid–base disorders of meta- throughout this wide range, both manufacturers use a double- bolic or respiratory origin and in the management of urinary indicator system of methyl red and bromothymol blue. Methyl conditions that require the urine to be maintained at a specific red produces a color change from red to yellow in the pH range pH. In respiratory or metabolic acidosis not related to renal 4 to 6, and bromothymol blue turns from yellow to blue in the function disorders, the urine is acidic; conversely, if respiratory range of 6 to 9. Therefore, in the pH range 5 to 9 measured by or metabolic alkalosis is present, the urine is alkaline. There- the reagent strips, colors progress from orange at pH 5 through fore, a urinary pH that does not conform to this pattern may yellow and green to a final deep blue at pH 9. be used to rule out the suspected condition, or, as discussed in Chapter 4, it may indicate a disorder resulting from the Methyl red + H+ → bromothymol blue – H+ kidneys’ inability to secrete or to reabsorb acid or base. (Red-orange → yellow) (green → blue) The precipitation of inorganic chemicals dissolved in the urine forms urinary crystals and renal calculi. This precip- No known substances interfere with urinary pH measure- itation depends on urinary pH and can be controlled by ments performed by reagent strips. However, bacterial growth maintaining the urine at a pH that is incompatible with the by certain organisms in a specimen may cause a marked alka- precipitation of the particular chemicals causing the calculi for- line shift, usually because of urea conversion to ammonia.2 mation. For example, calcium oxalate, a frequent constituent of renal calculi, precipitates primarily in acidic and not alkaline urine. Therefore, maintaining urine at an alkaline pH discour- Technical Tip 6-2. Care must be taken to prevent runover between the pH testing area and the adja- ages formation of the calculi. Knowledge of urinary pH is im- cent, highly acidic protein testing area on Multistix, as portant in the identification of crystals observed during this may produce a reading that is falsely acidic in an microscopic examination of the urine sediment. This will be alkaline urine. discussed in detail in Chapter 7. Maintaining an acidic urine can be valuable in treating UTIs caused by urea-splitting organisms because they do not multiply as readily in an acidic medium. These same organisms Protein are also responsible for the highly alkaline pH found in speci- mens that have been allowed to sit unpreserved for extended Of the routine chemical tests performed on urine, the most in- periods. Urinary pH is controlled primarily by dietary regula- dicative of renal disease is the protein determination. Often tion, although medications also may be used. People on high- proteinuria is associated with early renal disease, making the protein and high-meat diets tend to produce acidic urine, urinary protein test an important part of any physical exami- whereas urine from vegetarians is more alkaline due to the nation. Normal urine contains very little protein: usually, less 144 Part Two | Urinalysis of proteinuria are varied and can be grouped into three major SUMMARY 62 Clinical Significance categories: prerenal, renal, and postrenal, based on the origin of Urine pH of the protein. Respiratory or metabolic acidosis/ketosis Prerenal Proteinuria Respiratory or metabolic alkalosis As the name implies, prerenal proteinuria is caused by condi- Defects in renal tubular secretion and reabsorption of tions affecting the plasma before it reaches the kidney and there- acids and bases—renal tubular acidosis fore is not indicative of actual renal disease. Often this condition Renal calculi formation and prevention is transient and caused by increased levels of low-molecular- Treatment of UTIs weight plasma proteins, such as hemoglobin, myoglobin, and Precipitation/identification of crystals the acute-phase reactants associated with infection and inflam- mation. The increased filtration of these proteins exceeds the Determination of unsatisfactory specimens normal reabsorptive capacity of the renal tubules, resulting in an overflow of the proteins into the urine. Because reagent strips detect primarily albumin, prerenal proteinuria is usually not discovered in a routine urinalysis. SUMMARY 63 pH Reagent Strip Bence Jones Protein Reagents Methyl red, bromothymol blue Sensitivity Multistix: 5.0–8.5 in 0.5 increments A primary example of proteinuria due to increased levels of visually; 5.0–9 instrumentally serum protein is the excretion of Bence Jones protein by peo- ple with multiple myeloma, a proliferative disorder of the Chemstrip: 5.0–9.0 in 1.0 increments immunoglobulin-producing plasma cells. In multiple myeloma, Sources of error/ No known interfering substances the serum contains levels of monoclonal immunoglobulin light interference Runover from adjacent pads chains (Bence Jones protein) that are markedly elevated. This Old specimens low-molecular-weight protein is filtered in quantities exceeding the tubular reabsorption capacity and therefore is excreted in Bacterial growth the urine. Suspected cases of multiple myeloma must be diag- Correlations with Nitrite nosed by performing serum electrophoresis and immunoelec- other tests Leukocytes trophoresis. The screening test for Bence Jones protein is not Microscopic performed routinely, as cases of multiple myeloma are easily de- tected by chemical methods (see the Historical Note, Screening Test for Bence Jones Protein). than 10 mg/dL or 100 mg per 24 hours is excreted. This pro- Renal Proteinuria tein consists primarily of low-molecular-weight serum proteins that have been filtered by the glomerulus, as well as proteins Proteinuria associated with true renal disease may be the result produced in the genitourinary tract. Due to its low molecular of damage to the glomerular membrane or tubular dysfunction. weight, albumin is the major serum protein found in normal Glomerular Proteinuria urine. Even though it is present in high concentrations in the plasma, the normal albumin content in urine is low because the When the glomerular membrane is damaged, selective filtra- majority of albumin presented to the glomerulus is not filtered, tion is impaired and increased amounts of serum protein and, and much of the filtered albumin is reabsorbed by the tubules. Other proteins include small amounts of serum and tubular microglobulins; Tamm-Horsfall protein (THP), also known as HISTORICAL NOTE uromodulin, produced by the renal tubular epithelial cells; and proteins from prostatic, seminal, and vaginal secretions. Screening Test for Bence Jones Protein Uromodulin is a more recent name for THP. Uromodulin, a glycoprotein, is produced routinely in the ascending loop of Unlike other proteins, which coagulate and remain coagu- Henle.4 As will be discussed in Chapter 7, uromodulin forms lated when exposed to heat, Bence Jones protein coagulates the matrix of casts. at temperatures between 40°C and 60°C and dissolves when Clinical Significance the temperature reaches 100°C. Therefore, a specimen that appears turbid between 40°C and 60°C and clear at 100°C Demonstration of proteinuria in a routine analysis does not can be suspected of containing Bence Jones protein. Inter- always signify renal disease; however, its presence does require ference due to other precipitated proteins can be removed additional testing to determine whether the protein represents by filtering the specimen at 100°C and then observing the a condition that is normal or pathological. Clinical proteinuria specimen for turbidity as it cools to between 40°C and 60°C. is indicated at 30 mg/dL or greater (300 mg/L).2,5 The causes  Chapter 6 | Chemical Examination of Urine 145 eventually, red and white blood cells pass through the mem- believed to account for this condition. Patients suspected of brane and are excreted in the urine. Conditions that present orthostatic proteinuria are requested to empty the bladder the glomerular membrane with abnormal substances (e.g., before going to bed, collect a specimen immediately upon aris- amyloid material, toxic substances, and immune complexes ing in the morning, and collect a second specimen after found in lupus erythematosus and streptococcal glomeru- remaining in a vertical position for several hours. Both speci- lonephritis) are major causes of proteinuria due to glomerular mens are tested for protein, and if orthostatic proteinuria is damage. present, a negative reading will be seen on the first morning Increased pressure from the blood entering the glomerulus specimen, and a positive result will be found on the second may override the selective filtration of the glomerulus, causing specimen. increased albumin to enter the filtrate. This condition may be reversible, such as occurs during strenuous exercise and dehy- Tubular Proteinuria dration or is associated with hypertension. Proteinuria that Increased albumin is also present in disorders affecting tubular occurs during the latter months of pregnancy may indicate a reabsorption because the albumin that is normally filtered can preeclamptic state and should be considered by the physician no longer be reabsorbed. Other low-molecular-weight proteins in conjunction with other clinical symptoms, such as hyper- that are usually reabsorbed are also present. Causes of tubular tension, to determine whether this condition exists. dysfunction include exposure to toxic substances and heavy The discovery of protein, particularly in a random speci- metals, severe viral infections, and Fanconi syndrome. The men, is not always of pathological significance because several amount of protein that appears in the urine after glomerular benign causes of renal proteinuria exist. Benign proteinuria damage ranges from slightly above normal to 4 g/day, whereas is usually transient and can be produced by conditions such protein levels that are markedly elevated are seldom seen in as strenuous exercise, high fever, dehydration, and exposure tubular disorders. to cold. Postrenal Proteinuria Microalbuminuria Protein can be added to a urine specimen as it passes through The development of diabetic nephropathy leading to reduced the structures of the lower urinary tract (ureters, bladder, ure- glomerular filtration and eventual renal failure is a common thra, prostate, and vagina). Bacterial and fungal infections and occurrence in people with both type 1 and type 2 diabetes inflammations produce exudates containing protein from the mellitus. Onset of renal complications can be predicted interstitial fluid. The presence of blood as the result of injury first by detection of microalbuminuria (albumin levels in or menstrual contamination contributes protein, as does the the urine are 20 to 200 mg/L), and the progression of renal presence of prostatic fluid and large amounts of spermatozoa. disease can be prevented through better stabilization of blood glucose levels and control of hypertension. The presence of microalbuminuria also is associated with an increased risk of SUMMARY 64 Clinical Significance cardiovascular disease.6,7 of Urine Protein Orthostatic (Postural) Proteinuria Prerenal Tubular Disorders A persistent benign proteinuria occurs frequently in young Intravascular hemolysis Fanconi syndrome adults and is termed orthostatic proteinuria, or postural pro- teinuria. It occurs after periods spent in a vertical posture and Muscle injury Toxic agents/heavy metals disappears when a horizontal position is assumed. Increased Acute-phase reactants Severe viral infections pressure on the renal vein when in the vertical position is Multiple myeloma Renal Postrenal HISTORICAL NOTE Glomerular disorders Lower urinary tract infections/ inflammation Microalbuminuria Testing Immune complex Injury/trauma disorders Amyloidosis Menstrual contamination Before the development of current reagent strip methods Toxic agents Prostatic fluid/spermatozoa that are specific for albumin, detection of microalbumin- uria required collection of a 24-hour urine specimen. Diabetic nephropathy Vaginal secretions Specimens were tested using quantitative procedures for Strenuous exercise albumin. Results were reported in mg of albumin/24 hours Dehydration or as the albumin excretion rate (AER) in µg/min. With Hypertension these methods, microalbumin was considered significant when 30 to 300 mg of albumin were excreted in 24 hours Preeclampsia or the AER was 20 to 200 µg/min. Orthostatic or postural proteinuria 146 Part Two | Urinalysis Reagent Strip Reactions PROCEDURE 6-2 Traditional reagent strip testing for protein uses the principle of Sulfosalicylic Acid Precipitation Test the protein error of indicators to produce a visible colorimetric reaction. Contrary to the general belief that indicators produce Procedure specific colors in response to particular pH levels, certain indi- 1. Add 3 mL of 3% SSA reagent to 3 mL of centrifuged cators change color in the presence of protein even though the urine. pH of the medium remains constant. This is because protein 2. Mix by inversion, and observe for cloudiness. (primarily albumin) accepts hydrogen ions from the indicator. 3. Grade the degree of turbidity (see the following The test is more sensitive to albumin because albumin contains table). more amino groups to accept the hydrogen ions than other proteins. Depending on the manufacturer, the protein area of the strip contains either tetrabromophenol blue (Multistix) or 3′,3″,5′,5″-tetrachlorophenol, 3,4,5,6-tetrabromosulfonphthalein (Chemstrip) and an acid buffer to maintain the pH at a con- stant level. At a pH level of 3, both indicators appear yellow in the absence of protein; however, as the protein 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. Reporting of trace values may be a laboratory option. pH 3.0 Indicator + protein protein + H+ (Yellow) indicator – H+ (blue-green) Table Reporting SSA Turbidity Reaction Interference Protein Range Grade Turbidity (mg/dL) The major source of error with reagent strips occurs with highly buffered alkaline urine that overrides the acid buffer sys- Negative No increase in Less than 6 tem, producing a rise in pH and a color change unrelated to turbidity protein concentration. Likewise, a technical error of allowing Trace Noticeable turbidity 6–30 the reagent pad to remain in contact with the urine for a pro- 1+ Distinct turbidity, 30–100 longed period may remove the buffer. False-positive readings no granulation are obtained when the reaction does not take place under acidic conditions. Highly pigmented urine and contamination 2+ Turbidity, granulation, 100–200 of the container with quaternary ammonium compounds, no flocculation detergents, and antiseptics also cause false-positive readings. 3+ Turbidity, granulation, 200–400 A urine that is visibly bloody may cause results that are falsely flocculation elevated.2 A false-positive trace reading may occur in speci- 4+ Clumps of protein Greater mens with a high specific gravity. than 400 Sulfosalicylic Acid Precipitation Test The sulfosalicylic acid (SSA) test is a cold precipitation test that reacts equally with all forms of protein. Various con- Technical Tip 6-3. The specific gravity of the urine centrations and amounts of SSA can be used to precipitate specimen should be considered in evaluating urine protein, and methods vary greatly among laboratories. All protein, because a trace protein in a dilute specimen is precipitation tests must be performed on centrifuged speci- more significant than in a concentrated specimen. mens to remove any extraneous contamination. Based on the protocol of the laboratory, an SSA test may be performed as Testing for Microalbuminuria a confirmatory test in certain situations. This test may not be relevant to current laboratory practice; however, the proce- Several semiquantitative reagent strip methods have been de- dure is included in this section to serve as a reference if veloped so that patients at risk for renal disease can be moni- needed (Procedure 6-2).3 tored using random or first morning specimens. These methods  Chapter 6 | Chemical Examination of Urine 147 are based on immunochemical assays for albumin or albumin- SUMMARY 65 Protein Reagent Strip specific reagent strips that also measure creatinine to produce an albumin:creatinine (A:C) ratio. Reagents Multistix: Tetrabromophenol blue Immunochemical assays include the Micral-Test (Roche Chemstrip: 3’,3”,5’,5”-tetrachlorophenol Diagnostics, Indianapolis, IN) and the ImmunoDip (Sekisui Diagnostics, Framingham, MA). Both reagent strips are read 3,4,5,6-tetrabromosulfophthalein visually, and first morning specimens are recommended. Sensitivity Multistix: 15 to 30 mg/dL albumin Micral-Test reagent strips contain a gold-labeled antihu- Chemstrip: 6 mg/dL albumin man albumin antibody–enzyme conjugate. Strips are dipped Sources of error/ False positive into the urine up to a level marked on the strip and held for interference: Highly buffered interference alkaline 5 seconds. Albumin in the urine binds to the antibody. The bound and unbound conjugates move up the strip by wicking urine action. Unbound conjugates are removed in a captive zone by Pigmented specimens, phenazopyridine combining with albumin embedded in the strip. The urine Quaternary ammonium compounds albumin–bound conjugates continue up the strip and reach an (detergents) area containing enzyme substrate. The conjugated enzyme Antiseptics, chlorhexidine reacts with the substrate, producing colors ranging from white to red. The amount of color produced represents the amount Loss of buffer from prolonged exposure of albumin present in the urine. The color is compared with a of the strip to the specimen reagent chart on the reagent strip bottle after 1 minute. Results range High specific gravity from 0 to 10 mg/dL. False negative The ImmunoDip reagent strip uses an immunochromo- Proteins other than albumin graphic technique. Strips are packaged individually in spe- cially designed containers. The container is placed in the urine Microalbuminuria specimen for 3 minutes. A controlled amount of urine enters Correlations Blood the container through a vent hole. The urine encounters blue with other Nitrite latex particles coated with antihuman albumin antibody. Al- tests: Leukocytes bumin in the urine binds with the coated particles. The bound and unbound particles continue to migrate up the strip. The Microscopic migration is controlled by the size of the particles; unbound particles do not migrate as far as the bound particles. First a blue band is formed by the unbound particles. The bound particles continue to migrate and form a second blue band far- Reagent Strip Reactions ther up the strip. Therefore, the top band represents the bound particles (urine albumin), and the bottom band repre- Albumin sents unbound particles. The color intensity of the bands is Albumin reagent strips use the dye bis(3’,3”-diiodo-4’, compared against the manufacturer’s color chart. A darker bot- 4”-dihydroxy-5’,5”-dinitrophenyl)-3,4,5,6-tetrabromo sulphon- tom band represents less than 1.2 mg/dL, equal band colors phthalein (DIDNTB), which has a higher sensitivity and speci- represent 1.2 to 1.8 mg/dL, and a darker top band represents ficity for albumin. Whereas conventional protein reagent pads 2.0 to 8.0 mg/dL of albumin. A darker bottom band is nega- have a sensitivity of 30 mg/dL or greater and may include proteins tive, equal band color is borderline, and a darker top band other than albumin, the DIDNTB strips can measure albumin be- represents positive results. tween 8 and 15 mg/dL (80 and 150 mg/L) without inclusion of other proteins. Reaction interference by highly buffered alkaline Albumin:Creatinine Ratio urine (always a concern with conventional reagent strips) is con- trolled by using paper treated with bis-(heptapropylene glycol) The Clinitek Microalbumin reagent strips and the Multistix Pro carbonate. The addition of polymethyl vinyl ether decreases reagent strips (Siemens Healthcare Diagnostics, Inc., Tarrytown, the nonspecific binding of polyamino acids to the albumin pad. NY) provide simultaneous measurement of albumin/protein Colors range from pale green to aqua blue. Falsely elevated results and creatinine that permits an estimation of the 24-hour can be caused by urine that is visibly bloody, whereas urine that microalbumin excretion.8 As discussed in Chapter 4, creatinine is abnormally colored may interfere with the readings.2 is produced and excreted at a consistent rate for each individ- ual. Therefore, by comparing the albumin excretion to the cre- Creatinine atinine excretion, the albumin reading can be corrected for The principle of the reagent strip for creatinine is based on the either overhydration or dehydration in a random specimen. In pseudoperoxidase activity of copper–creatinine complexes. The addition to including creatinine on the reagent strip, the albu- reaction follows the same principle as the reaction for blood on min low-test pad is changed to a dye-binding reaction that is the reagent strips discussed later in this chapter. Reagent strips more specific for albumin than the protein error of indicators’ contain copper sulfate (CuSO4), 3,3’,5,5’-tetramethylbenzidine reaction on strips measuring protein. (TMB), and diisopropyl benzene dihydroperoxide (DBDH). 148 Part Two | Urinalysis Creatinine in the urine combines with the copper sulfate to these strips. The strips can be read manually or on automated form copper–creatinine peroxidase. This reacts with the perox- Clinitek instruments. The protein-high reaction uses the pro- ide DBDH, releasing oxygen ions that oxidize the chromogen tein error of indicators principle, whereas the protein-low TMB and producing a color change from orange through green reaction is the dye-binding method discussed previously. to blue.5 Results are reported as the protein:creatinine ratio, although the protein-low result is used in the calculation. Results from CuSO4 + CRE → Cu(CRE) peroxidase the Clinitek are calculated automatically. Results are reported Cu(CRE) peroxidase as normal or abnormal. A result of normal dilute indicates that DBDH + TMB oxidized TMB + H2O the specimen should be re-collected, making sure it is a first (peroxidase) (chromogen) (orange to blue) morning specimen. When the reagent strip is read manually, a manufacturer- Results are reported as 10, 50, 100, 200, or 300 mg/dL or supplied chart is used to determine the ratio based on the results 0.9, 4.4, 8.8, 17.7, or 26.5 mmol/L of creatinine. of the readings for protein-high, protein-low, and creatinine. Reagent strips are unable to detect the absence of creati- When using this chart, the higher of the protein-low or protein- nine. Results that are falsely elevated can be caused by urine high result is used (Fig. 6-3).9 that is visibly bloody, as well as the presence of the gastric acid– reducing medication cimetidine (Tagamet). Urine that is abnormally colored also may interfere with the readings. Glucose No creatinine readings are considered abnormal, as creati- Because of its value in the detection and monitoring of nine is normally present in concentrations of 10 to 300 mg/dL. diabetes mellitus, the glucose test is the chemical analysis The purpose of the creatinine measurement is to correlate the performed most frequently on urine. Due to the nonspecific albumin concentration to the urine concentration, producing a symptoms associated with the onset of diabetes, it is esti- semiquantitative albumin:creatinine ratio (A:C) ratio. mated that more than half of the cases in the world are undi- Albumin/Protein:Creatinine Ratio agnosed. Therefore, blood and urine glucose tests are included Both automated and manual methods are available for determin- in all physical examinations and are often the focus of mass ing the A:C ratio based on the reactions discussed previously. health screening programs. Early diagnosis of diabetes melli- The Clinitek Microalbumin reagent strips are designed for in- tus through blood and urine glucose tests provides a prog- strumental use only. Strips are read on Clinitek Urine Chemistry nosis that is greatly improved. Using reagent strip methods Analyzers. The strips measure only albumin and creatinine, and for both blood and urine glucose testing that are currently the analyzer calculates the A:C ratio automatically. Results are available, patients can monitor themselves at home and can displayed and printed out for albumin, creatinine, and the A:C detect regulatory problems before the development of serious ratio in both conventional and SI units. Abnormal results for the complications. A:C ratio are 30 to 300 mg/g or 3.4 to 33.9 mg/mmol.8 The Siemens Multistix Pro 10 reagent strips include reagent Clinical Significance pads for creatinine, protein-high, and protein-low (albumin), Under normal circumstances, almost all the glucose filtered by along with pads for glucose, ketones, blood, nitrite, LE, pH, the glomerulus is actively reabsorbed in the proximal convoluted bilirubin, and specific gravity. Urobilinogen is not included on tubule; therefore, urine contains only minute amounts of glucose. SUMMARY 66 Microalbumin Testing Immunological Tests Albumin:Creatinine Ratio Micral-Test ImmunoDip Clinitest Microalbumin Strips/Multistix-Pro Principle Enzyme immunoassay Immunochromographics Sensitive albumin tests related to creatinine concentration to correct for patient hydration Sensitivity 0–10 mg/dL 1.2–8.0 mg/dL Albumin: 10–150 mg/L Creatinine: 10–300 mg/dL, 0.9–26.5 mmol/L Reagents Gold-labeled antibody Antibody-coated blue Albumin: dye DIDNTB B-galactosidase latex particles Creatinine: CuSO4, 3,3’,5,5’-TMB, and DBDH Chlorophenol red galactoside Interference False negative: Dilute False negative: Dilute Visibly bloody or abnormally colored urine urine urine Creatinine: cimetidine: False positive  Chapter 6 | Chemical Examination of Urine 149 Reported Creatinine result increased levels of circulating glucose. Epinephrine is also a protein result (mg/dL) strong inhibitor of insulin secretion and is increased when the (mg/dL) 10 50 100 200 300 body is subjected to severe stress, which accounts for the Negative Recollect* No glucosuria seen in conjunction with cerebrovascular trauma rm and myocardial infarction. al 15 Ab Glycosuria occurs in the absence of hyperglycemia when no the reabsorption of glucose by the renal tubules is compro- 30 rm 100, 300, al mised. This is frequently referred to as “renal glycosuria” and or 2000 is seen in end-stage renal disease, cystinosis, and Fanconi syn- *Specimen is too dilute to determine ratio result accurately. Repeat test on new drome. Glycosuria not associated with gestational diabetes is specimen, preferably a first-morning collection. seen occasionally as a result of a temporary lowering of the Figure 6–3 A protein-creatinine ratio determination chart. (Image renal threshold for glucose during pregnancy. adapted from Bayer HealthCare LLC, Elkhart, IN.) Reagent Strip (Glucose Oxidase) Reaction The glucose oxidase procedure provides a specific test for glu- Tubular reabsorption of glucose is by active transport in response cose. Reagent strips employ the glucose oxidase testing method to the body’s need to maintain an adequate concentration of by impregnating the testing area with a mixture of glucose ox- glucose. Should the blood level of glucose become elevated idase, peroxidase, chromogen, and buffer to produce a double (hyperglycemia), as occurs in diabetes mellitus, the tubular sequential enzyme reaction. In the first step, glucose oxidase transport of glucose has reached its renal threshold, and glucose catalyzes a reaction between glucose and room air (oxygen) to appears in the urine. The blood level at which tubular reabsorp- produce gluconic acid and peroxide. In the second step, per- tion stops (renal threshold) for glucose is approximately 160 to oxidase catalyzes the reaction between peroxide and chro- 180 mg/dL. Blood glucose levels fluctuate, and a typical person mogen to form an oxidized colored compound that is who is nonfasting may have glycosuria after a meal containing a produced in direct proportion to the concentration of glucose. high glucose content. Therefore, the most informative glucose re- sults are obtained from specimens collected under controlled Glucose oxidase 1. Glucose + O2 (air) gluconic acid + H2O2 conditions. Fasting before the collection of specimens for screen- ing tests is recommended. For purposes of diabetes monitoring, Peroxidase 2. H2O2 + chromogen oxidized specimens are usually tested 2 hours after meals. A first morning colored chromogen + H2O specimen does not always represent a fasting specimen because glucose from an evening meal may remain in the bladder Reagent strip manufacturers use several different chro- overnight, and patients should be advised to empty the bladder mogens, including potassium iodide (green to brown) (Multistix) and collect the second specimen.2 and tetramethylbenzidine (yellow to green) (Chemstrip). Urine Hyperglycemia that occurs during pregnancy and disap- glucose may be reported in terms of negative, trace, 1+, 2+, 3+, pears after delivery is called gestational diabetes. The onset of and 4+; however, the color charts also provide semiquantitative hyperglycemia and glycosuria is normally around the sixth measurements ranging from 100 mg/dL to 2 g/dL or 0.1% to 2%. month of pregnancy, although glycosuria may occur sooner. The American Diabetes Association recommends semiquantita- Hormones secreted by the placenta block the action of insulin, tive reporting. resulting in insulin resistance and hyperglycemia. Detection of gestational diabetes is important to the welfare of both the mother and baby because glucose crosses the placenta, whereas insulin does not. The baby develops high glucose levels, causing SUMMARY 67 Clinical Significance the baby’s pancreas to produce more insulin. The excess glucose of Urine Glucose transferred to the baby is stored as fat, resulting in a large baby Hyperglycemia-Associated Renal-Associated (macrosomia) at risk for obesity and, later, type 2 diabetes. Women who have gestational diabetes also are prone to devel- Diabetes mellitus Fanconi syndrome oping type 2 diabetes mellitus in later years. Pancreatitis Advanced renal disease Hyperglycemia of nondiabetic origin is seen in a variety of Pancreatic cancer Osteomalacia disorders and also produces glycosuria. Many of these disorders are associated with hormonal function and include pancreatitis, Acromegaly Pregnancy acromegaly, Cushing syndrome, hyperthyroidism, pheochromo- Cushing syndrome cytoma, and thyrotoxicosis. The hormones glucagon, epinephrine, Hyperthyroidism cortisol, thyroxine, and growth hormone, which are increased Pheochromocytoma in these disorders, work in opposition to insulin, thereby pro- ducing hyperglycemia and glucosuria. Whereas a primary Central nervous system damage function of insulin is to convert glucose to glycogen for storage Stress (glycogenesis), these opposing hormones cause the break- Gestational diabetes down of glycogen to glucose (glycogenolysis), resulting in 150 Part Two | Urinalysis Reaction Interference SUMMARY 68 Glucose Reagent Strip Because the glucose oxidase method is specific for glucose, Reagents Multistix false-positive reactions are not obtained from other urinary con- stituents, including reducing sugars that may be present. False- Glucose oxidase positive reactions may occur, however, if containers become Peroxidase contaminated with peroxide or strong oxidizing detergents from Potassium iodide disinfectants used on laboratory instruments. Chemstrip Substances that interfere with the enzymatic reaction or strong reducing agents, such as ascorbic acid, that prevent Glucose oxidase oxidation of the chromogen may produce false-negative re- Peroxidase sults. To minimize interference from ascorbic acid, reagent Tetramethylbenzidine strip manufacturers are incorporating additional chemicals Sensitivity Multistix: 75 to 125 mg/dL into the test pads. An example is iodate that oxidizes ascorbic acid so that it cannot interfere with the oxidation of the chro- Chemstrip: 40 mg/dL mogen. Product literature should be reviewed carefully for Interference False positive current information regarding all interfering substances. High Contamination by oxidizing levels of ketones also affect glucose oxidase tests at low glucose agents and detergents concentrations; however, because high levels of ketones are False negative usually accompanied by marked glycosuria, this seldom pres- ents a problem. High specific gravity and low temperature High levels of ascorbic acid may decrease the sensitivity of the test. By far, the greatest High levels of ketones source of false-negative glucose results is the technical error High specific gravity of allowing specimens to remain unpreserved at room tem- Low temperatures perature for extended periods, subjecting the glucose to bacterial degradation. Improperly preserved specimens Correlations Ketones Copper Reduction Test (Clinitest) with other tests Protein Measurement of glucose by the copper reduction method was one of the earliest chemical tests performed on urine. The test relies on the ability of glucose and other substances to reduce copper sulfate to cuprous oxide in the presence of alkali and gently, and the color, ranging from blue to orange/red, can be heat. A color change progressing from a negative blue (CuSO4) compared with the manufacturer’s color chart to determine the through green, yellow, and orange/red (Cu2O) occurs when the approximate amount of reducing substance. reaction takes place. Care must be taken to observe the reaction closely as it is taking place because at high glucose levels, a phenomenon Heat CuSO4 (cupric sulfide) + reducing substance known as “pass through” may occur. When this happens, Alkali the color produced passes through the orange/red stage and Cu2O (cuprous oxide) + oxidized substance → color returns to a green-brown color, and if not observed, a high (blue/green → orange/red) glucose level may be reported as negative. An alternative method using two drops instead of five drops of urine can The classic Benedict solution was developed in 1908 and minimize the occurrence of “pass through.” A separate color contained copper sulfate, sodium carbonate, and sodium citrate chart must be used to interpret the reaction. This chart pro- buffer.10 Urine was added to the solution, heat was applied, and vides values up to 5 g/dL, whereas the five-drop method is the resulting precipitate was observed for color. A more con- limited to 2 g/dL. venient method that employs Benedict’s principle is the Clinitest The sensitivity of Clinitest to glucose is reduced to a mini- tablet (Siemens Healthcare Diagnostics, Inc., Tarrytown, NY). mum of 200 mg/dL, so the Clinitest cannot be used as a confir- The tablets contain copper sulfate, sodium carbonate, sodium matory test for glucose. As a nonspecific test for reducing citrate, and sodium hydroxide. Upon addition of the tablet to substances, Clinitest is subject to interference from other reducing water and urine, heat is produced by the hydrolysis of sodium sugars, including galactose, lactose, fructose, maltose, pentose, hydroxide and its reaction with sodium citrate, and carbon ascorbic acid, certain drug metabolites, and antibiotics, such as dioxide is released from the sodium carbonate to prevent room the cephalosporins. air from interfering with the reduction reaction. Thick-walled Clinitest tablets are very hygroscopic and should be stored tubes should be placed in a heat-resistant rack and not held in in their original, tightly closed packages. A strong blue color the hand because the reaction heat could cause a burn. At the in the unused tablets suggests deterioration due to moisture conclusion of the effervescent reaction, the tube is shaken accumulation, as does vigorous tablet fizzing.  Chapter 6 | Chemical Examination of Urine 151 Clinical Significance of Clinitest Ketones In addition to glucose, commonly found reducing sugars The term “ketones” represents three intermediate products of include galactose, fructose, pentose, and lactose, of which fat metabolism, namely, acetone (2%), acetoacetic acid (20%), galactose is the most clinically significant. Galactose in the and β-hydroxybutyrate (78%). Normally, measurable amounts urine of a newborn represents an “inborn error of metabo- of ketones do not appear in the urine because all the metabo- lism” in which a lack of the enzyme galactose-1-phosphate lized fat is completely broken down into carbon dioxide and uridyl transferase (GALT) prevents breakdown of ingested water. However, when the use of available carbohydrate as the galactose and results in failure to thrive and other complica- major source of energy becomes compromised, body stores of tions, including death. Depending on the laboratory popu- fat must be metabolized to supply energy. Then ketones are lation, the Clinitest was performed on all pediatric specimens detected in urine. from patients up to at least the age of 2 years. However, now all states have incorporated screening for galactosemia into Clinical Significance their required newborn screening programs (see Chapter 9) because early detection followed by dietary restriction can Clinical reasons for increased fat metabolism include the control the condition. Because of the increased sensitivity inability to metabolize carbohydrate, as occurs in diabetes and specificity of the newborn blood screening test for the mellitus; increased loss of carbohydrate from vomiting; and activity of GALT, the urine screening for carbohydrate me- inadequate intake of carbohydrate associated with starvation tabolism disorders using the Clinitest reagent tablets is no and malabsorption. longer recommended.11 The appearance of other reducing Testing for urinary ketones is most valuable in the sugars is usually of minimal clinical significance, and lactose management and monitoring of insulin-dependent (type 1) is frequently found in the urine of nursing mothers. diabetes mellitus. Ketonuria shows a deficiency in insulin, indicating the need to regulate dosage. It is often an early indicator of insufficient insulin dosage in type 1 diabetes and in patients who experience medical problems in addition to diabetes. Increased accumulation of ketones in the blood leads Technical Tip 6-4. Keep in mind that table sugar is su- to electrolyte imbalance, dehydration, and, if not corrected, crose, a nonreducing sugar, which does not react with acidosis and eventual diabetic coma. Clinitest or glucose oxidase strips. Therefore, it cannot The use of multiple-test strips in hospital laboratories be used as a control or in preparation of a laboratory often produces positive ketone tests unrelated to diabetes exercise for glucose testing. because the patient’s illness either prevents adequate intake or absorption of carbohydrates or produces an accelerated loss, as in the case of vomiting. Clinics for weight loss and eating disorders can use a practical application of ketonuria PROCEDURE 6-3 produced by avoidance of carbohydrates to monitor patients. Frequent strenuous exercise can cause overuse of available Clinitest Procedure carbohydrates and produce ketonuria that can be toxic to the Procedure kidney tubules. 1. Place a thick glass test tube in a rack; add five drops of urine. 2. Add 10 drops of distilled water to the urine in the test tube. SUMMARY 69 Clinical Significance 3. Drop one Clinitest tablet into the test tube, and of Urine Ketones observe the reaction until completion (cessation of Diabetic acidosis boiling). Insulin dosage monitoring CAUTION: The reaction mixture gets very hot. Do not touch the bottom area of the test tube. Use a Starvation thick glass test tube only. Malabsorption/pancreatic disorders 4. Wait 15 seconds after boiling has stopped, and gently Cold exposure shake the contents of the tube. Strenuous exercise 5. Compare the color of the mixture to the Clinitest Vomiting color chart, and record the result in mg/dL or per- Inborn errors of amino acid metabolism (see Chapter 9) cent. Observe for the possibility of the “pass through” phenomenon. If present, repeat the procedure using Alcoholism two drops of urine instead of five drops. Febrile state in children 152 Part Two | Urinalysis Reagent Strip Reactions SUMMARY 610 Ketone Reagent Strip The three ketone compounds are not present in equal amounts Reagents Sodium nitroprusside in urine. Both acetone and β-hydroxybutyric acid are produced from acetoacetic acid (Fig. 6-4). The proportions of 78% Glycine (Chemstrip) β-hydroxybutyric acid, 20% acetoacetic acid, and 2% acetone Sensitivity Multistix: 5–10 mg/dL are relatively constant in all specimens. acetoacetic acid Reagent strip tests use the sodium nitroprusside (nitrofer- Chemstrip: 9 mg/dL ace- ricyanide) reaction to measure ketones. In this reaction, ace- toacetic acid; 70 mg/dL toacetic acid in an alkaline medium reacts with sodium acetone nitroprusside to produce a purple color. The test does not Interference False positive: measure β-hydroxybutyrate and is only slightly sensitive to acetone when glycine is also present; however, inasmuch as these Phthalein dyes compounds are derived from acetoacetic acid, their presence can Highly pigmented red be assumed, and it is not necessary to perform individual tests. urine Results are reported qualitatively as negative, trace, small (1+), Levodopa moderate (2+), or large (3+), or semiquantitatively as negative, Medications containing trace (5 mg/dL), small (15 mg/dL), moderate (40 mg/dL), free sulfhydryl groups or large (80 to 160 mg/dL). False negative: Acetoacetate (and acetone) + sodium nitroprusside Improperly preserved alkaline specimens + (glycine) purple color Correlations Glucose Reaction Interference Large amounts of levodopa and medications containing sulfhydryl groups, including mercaptoethane sulfonate sodium and lactose in tablet form. The addition of lactose gives better (MESNA) and captopril, may produce atypical color reactions. color differentiation. Acetest tablets are hygroscopic; if the Reactions with interfering substances frequently fade on stand- specimen is not absorbed completely within 30 seconds, a new ing, whereas color development from acetoacetic acid in- tablet should be used. See Procedure 6-4. creases, which leads to false-positive results from readings that are improperly timed. Values that are falsely decreased due to the volatilization of acetone and the breakdown of acetoacetic acid by bacteria are seen in specimens that have been preserved Blood improperly. Blood may be present in the urine either in the form of intact The Acetest tablet test has been used as a confirmatory red blood cells (RBCs) (hematuria) or as the product of RBC test for questionable reagent strip results; however, it was destruction, hemoglobin (hemoglobinuria). As discussed in primarily used for testing serum and other bodily fluids and Chapter 5, blood present in large quantities can be detected dilutions of these fluids for severe ketosis. The Clinical and visually; hematuria produces a cloudy red urine, and hemo- Laboratory Standards Institute (CLSI) states that the confir- globinuria appears as a clear red specimen. Because any matory test for ketones using the Acetest may not be relevant amount of blood greater than five cells per microliter of urine to current laboratory practice.3 Currently, new methods meas- is considered clinically significant, visual examination cannot uring β-hydroxybutyrate using reagent strips have been de- be relied upon to detect the presence of blood. Microscopic veloped to provide automated methods for testing serum and examination of the urinary sediment shows intact RBCs, but other body fluids. Notice in Figure 6-4 the ketone with the free hemoglobin produced either by hemolytic disorders or highest concentration is β-hydroxybutyrate. lysis of RBCs is not detected. Therefore, chemical tests for he- moglobin provide the most accurate means for determining Acetest Tablets the presence of blood. Once blood has been detected, the mi- Acetest (Siemens Healthcare Diagnostics, Inc., Deerfield, IL) croscopic examination can be used to differentiate between provides sodium nitroprusside, glycine, disodium phosphate, hematuria and hemoglobinuria. OH O O +2H –CO2 CH3 C CH2 COOH CH3 C CH2 COOH CH3 C CH3 –2H H Figure 6–4 Production of acetone and butyrate from b-hydroxybutyrate Acetoacetic acid Acetone acetoacetic acid.  Chapter 6 | Chemical Examination of Urine 153 PROCEDURE 6-4 Myoglobinuria Myoglobin, a heme-containing protein found in muscle tissue, Acetest Procedure not only reacts positively with the reagent strip test for blood Procedure but also produces a clear red-brown urine. In myoglobinuria, 1. Remove the Acetest tablet from the bottle, and place the presence of myoglobin rather than hemoglobin should be it on a clean, dry piece of white paper. suspected in patients with conditions associated with muscle 2. Place one drop of urine on top of the tablet. destruction (rhabdomyolysis). Examples of these conditions include trauma, crush syndromes, prolonged coma, convul- 3. Wait 30 seconds. sions, muscle-wasting diseases, alcoholism, heroin abuse, and 4. Compare the tablet color with the manufacturer- extensive exertion. The development of rhabdomyolysis has supplied color chart. been found to be a side effect in certain patients taking the 5. A positive result is indicated by a purple color. cholesterol-lowering statin medications.12 The heme portion Report as negative, small, moderate, or large. of myoglobin is toxic to the renal tubules, and high concen- trations can cause acute renal failure. The massive hemoglo- binuria seen in hemolytic transfusion reactions also is associated with acute renal failure. Clinical Significance Reagent Strip Reactions The finding of a positive reagent strip test result for blood in- Chemical tests for blood use the pseudoperoxidase activity of dicates the presence of RBCs, hemoglobin, or myoglobin. Each hemoglobin to catalyze a reaction between the heme compo- of these has a different clinical significance. nent of both hemoglobin and myoglobin and the chromogen tetramethylbenzidine to produce an oxidized chromogen, Hematuria which has a green-blue color. Hematuria is most closely related to disorders of renal or gen- Hemoglobin itourinary origin in which bleeding is the result of trauma or H2O2 + chromogen oxidized chromogen + H2O Peroxidase damage to the organs of these systems. Major causes of hema- turia include renal calculi, glomerular diseases, tumors, trauma, pyelonephritis, exposure to toxic chemicals, and anti- Reagent strip manufacturers incorporate peroxide and coagulant therapy. The laboratory is frequently requested to tetramethylbenzidine into the blood testing area. Two color perform a urinalysis when patients presenting with severe back charts are provided that correspond to the reactions that occur and abdominal pain are suspected of having renal calculi. In such cases, hematuria is usually of a small to moderate degree, but its presence can be essential to the diagnosis. Hematuria SUMMARY 611 Clinical Significance of nonpathological significance is observed after strenuous of a Positive Reaction exercise and during menstruation. for Blood Hematuria Brown recluse spider Hemoglobinuria bites Renal calculi Hemoglobinuria may result from the lysis of RBCs produced Glomerulonephritis Myoglobinuria in the urinary tract, particularly in dilute, alkaline urine. It Muscular trauma Pyelonephritis also may result from intravascular hemolysis and the subse- Tumors Crush injuries quent filtering of hemoglobin through the glomerulus. Lysis of RBCs in the urine usually shows a mixture of hemoglo- Trauma Prolonged coma binuria and hematuria, whereas no RBCs are seen in cases Exposure to toxic chemicals Convulsions of intravascular hemolysis. Under normal conditions, the Muscle-wasting diseases Anticoagulants formation of large hemoglobin–haptoglobin complexes in Strenuous exercise Alcoholism/overdose the circulation prevents the glomerular filtration of hemo- globin. When the amount of free hemoglobin present Hemoglobinuria Drug abuse exceeds the haptoglobin content—as occurs in hemolytic Transfusion reactions Extensive exertion anemias, transfusion reactions, severe burns, bites from the Cholesterol-lowering Hemolytic anemias brown recluse spider, infections, and strenuous exercise— statin medications hemoglobin is available for glomerular filtration. Reabsorp- Severe burns tion of filtered hemoglobin also results in the appearance Infections/malaria of large yellow-brown granules of denatured ferritin called Strenuous exercise/ hemosiderin in the renal tubular epithelial cells and in the RBC trauma urine sediment. 154 Part Two | Urinalysis with hemoglobinuria, myoglobinuria, and hematuria (RBCs). SUMMARY 612 Blood Reagent Strip In the presence of free hemoglobin/myoglobin, a uniform color ranging from a negative yellow through green to a strongly pos- Reagents Multistix: Diisopropylbenzene itive green-blue appears on the pad. In contrast, intact RBCs dihydroperoxide and 3,3’,5,5’- are lysed when they come in contact with the pad, and the lib- tetramethylbenzidine erated hemoglobin produces an isolated reaction that results Chemstrip: dimethyldihydroperoxy- in a speckled pattern on the pad. Reagent strip tests can detect hexane and tetramethylbenzidine concentrations as low as five RBCs per microliter; however, care must be taken when comparing these figures with the ac- Sensitivity Multistix: 5–20 RBCs/mL, 0.015–0.062 tual microscopic values because the absorbent nature of the mg/dL hemoglobin pad attracts some urine. The terms trace, small, moderate, and Chemstrip: 5 RBCs/mL, hemoglobin large or trace, 1+, 2+, and 3+ are used for reporting. corresponding to 10 RBCs/mL Interference False positive: Reaction Interference Strong oxidizing agents False-positive reactions due to menstrual contamination may Bacterial peroxidases be seen. They also occur if strong oxidizing detergents, such as sodium hypochlorite (bleach), are present in the specimen Menstrual contamination container.2 Vegetable peroxidase and bacterial enzymes, includ- False negative: ing an Escherichia coli peroxidase, also may cause false-positive High specific gravity/crenated cells reactions. Therefore, sediments containing bacteria should be Formalin checked closely for the presence of RBCs. Traditionally, ascorbic acid (vitamin C) has been associated Captopril with false-negative reagent strip reactions for blood. Both Mul- High concentrations of nitrite tistix and Chemstrip have modified their reagent strips to reduce Ascorbic acid greater than 25 mg/dL this interference to very high levels (25 mg/dL) of ascorbic acid. Unmixed specimens Multistix uses a peroxide that is less subject to reduction by ascorbic acid, and Chemstrip overlays the reagent pad with an Correlations Protein iodate-impregnated mesh that oxidizes the ascorbic acid before with other Microscopic it reaches the reaction pad. False-negative reactions can result tests when urine with a high specific gravity contains crenated RBCs that do not lyse when they come in contact with the reagent pad. Decreased reactivity also may be seen when formalin is used as a preservative or when the hypertension medication captopril present. RBCs settle to the bottom of the specimen container, or high concentrations of nitrite