Urinalysis Chem Pt. 1 PDF

3920_Ch05_071-098 23/01/14 10:13 AM Page 72 72 Part Two | Urinalysis KEY TERMS Bacteriuria Hemosiderin Postrenal proteinuria Bilirubin Jaundice...

3920_Ch05_071-098 23/01/14 10:13 AM Page 72 72 Part Two | Urinalysis KEY TERMS Bacteriuria Hemosiderin Postrenal proteinuria Bilirubin Jaundice Prerenal proteinuria Fanconi syndrome Ketonuria Protein error of indicators Ferritin Leukocyturia Proteinuria Glycosuria Microalbuminuria Stercobilinogen Hematuria Myoglobinuria Urobilinogen Hemoglobinuria Orthostatic proteinuria Uromodulin Reagent Strips Errors Caused by Improper Technique 1. Formed elements such as red and white blood cells sink Routine chemical examination of urine has changed dramat- to the bottom of the specimen and will be undetected in ically since the early days of urine testing, due to the devel- an unmixed specimen. opment of the reagent strip method for chemical analysis. Reagent strips currently provide a simple, rapid means for 2. Allowing the strip to remain in the urine for an extended performing medically significant chemical analysis of urine, period may cause leaching of reagents from the pads. including pH, protein, glucose, ketones, blood, bilirubin, 3. Excess urine remaining on the strip after its removal urobilinogen, nitrite, leukocytes, and specific gravity. The from the specimen can produce a run-over between two major types of reagent strips are manufactured under chemicals on adjacent pads, producing distortion of the trade names Multistix (Siemens Healthcare Diagnostics, the colors. To ensure against run-over, blotting the edge Deerfield, IN) and Chemstrip (Roche Diagnostics, Indianapolis, of the strip on absorbent paper and holding the strip IN). These products are available with single-or multiple- horizontally while comparing it with the color chart is testing areas, and the brand and number of tests used are a mat- recommended. ter of laboratory preference. Certain variations relating to chem- 4. The timing for reactions to take place varies between ical reactions, sensitivity, specificity, and interfering substances tests and manufacturers, and ranges from an immediate occur among the products and are discussed in the following reaction for pH to 120 seconds for leukocyte esterase. sections. Reagent strip brands are also specified by instrumen- For the best semiquantitative results, the manufacturer’s tation manufacturers. stated time should be followed; however, when precise Reagent strips consist of chemical-impregnated absorbent timing cannot be achieved, manufacturers recommend pads attached to a plastic strip. A color-producing chemical re- that reactions be read between 60 and 120 seconds, with action takes place when the absorbent pad comes in contact the leukocyte esterase reaction read at 120 seconds. with urine. The reactions are interpreted by comparing the 5. A good light source is essential for accurate interpreta- color produced on the pad within the required time frame with tion of color reactions. a chart supplied by the manufacturer. Several colors or inten- sities of a color for each substance being tested appear on the chart. By careful comparison of the colors on the chart and the PROCEDURE 5-1 strip, a semiquantitative value of trace, 1+, 2+, 3+, or 4+ can be reported. An estimate of the milligrams per deciliter present Reagent Strip Technique1,2 is available for appropriate testing areas. Automated reagent 1. Dip the reagent strip briefly into a well-mixed uncen- strip readers also provide Système International units. trifuged urine specimen at room temperature. Reagent Strip Technique 2. Remove excess urine by touching the edge of the strip to the container as the strip is withdrawn. Testing methodology includes dipping the reagent strip com- 3. Blot the edge of the strip on a disposable absorbent pletely, but briefly, into a well-mixed specimen, removing ex- pad. cess urine from the strip by running the edge of the strip on the container when withdrawing it from the specimen, blotting 4. Wait the specified amount of time for the reaction to it horizontally on an absorbent medium, waiting the specified occur. length of time for reactions to take place, and comparing the 5. Compare the color reaction of the strip pads to the colored reactions against the manufacturer’s chart using a good manufacturer’s color chart in good lighting. light source. 3920_Ch05_071-098 23/01/14 10:13 AM Page 73 Chapter 5 | Chemical Examination of Urine 73 6. The strip must be held close to the color chart without reagent strip interference is the masking of color reactions by actually being placed on the chart. Automated reagent the orange pigment present in the urine of persons taking strip instruments standardize the color interpretation phenazopyridine compounds. If laboratory personnel do not and timing of the reaction and are not subject to room recognize the presence of this pigment or other pigments, they lighting deficiencies or inconsistency among laboratory will report many erroneous results. personnel (Appendix A). 7. Reagent strips and color charts from different manufac- Confirmatory Testing turers are not interchangeable. Confirmatory tests are defined as test using different reagents 8. Specimens that have been refrigerated must be allowed or methodologies to detect the same substances as detected to return to room temperature prior to reagent strip by the reagent strips with the same or greater sensitivity or testing, as the enzymatic reactions on the strips are tem- specificity.4 perature dependent. Nonreagent strip testing procedures using tablets and liquid chemicals may be available when questionable results Handling and Storing Reagent Strips are obtained or highly pigmented specimens are encountered. In the past, many of these procedures were used routinely to In addition to using correct testing technique, reagent strips confirm positive results. Increased specificity and sensitivity must be protected from deterioration caused by moisture, of reagent strips and the use of automated strip readers have volatile chemicals, heat, and light. Reagent strips are packaged reduced the need for routine use of these procedures.5,6 The in opaque containers with a desiccant to protect them from chemical reliability of these procedures also must be checked light and moisture. Strips are removed just prior to testing, and using positive and negative controls. the bottle is tightly resealed immediately. Bottles should not be Specific confirmatory tests are discussed in this chapter opened in the presence of volatile fumes. Manufacturers rec- under their specific sections or the Historical Notes devoted to ommend that reagent strips be stored at room temperature the chemical parameters for which they are used. The princi- below 30°C (but never refrigerated). All bottles are stamped ples and procedures for these tests are included to provide ad- with an expiration date that represents the functional life ex- ditional information on the principles of the reagent strips and pectancy of the chemical pads. Reagent strips must not be used to provide the methodology to perform these tests if necessary. past the expiration date. Care must be taken not to touch the Institutional protocol will determine the situations when they chemical pads when removing the strips. A visual inspection should be performed. 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. pH Quality Control of Reagent Strips Along with the lungs, the kidneys are the major regulators of Reagent strips must be checked with both positive and negative the acid–base content in the body. They do this through the controls a minimum of once every 24 hours. Many laboratories secretion of hydrogen in the form of ammonium ions, hydro- perform this check at the beginning of each shift. Testing is gen phosphate, and weak organic acids, and by the reabsorp- also performed when a new bottle of reagent strips is opened, tion of bicarbonate from the filtrate in the convoluted tubules questionable results are obtained, or there is concern about the (see Chapter 2). A healthy individual usually produces a first integrity of the strips. All quality control results must be morning specimen with a slightly acidic pH of 5.0 to 6.0; a recorded following laboratory protocol. Several companies more alkaline pH is found following meals (alkaline tide). The manufacture both positive and negative controls. Distilled pH of normal random samples can range from 4.5 to 8.0. Con- water is not recommended as a negative control because sequently, no normal values are assigned to urinary pH, and reagent strip chemical reactions are designed to perform at it must be considered in conjunction with other patient ionic concentrations similar to urine. All readings of the nega- information, such as the acid–base content of the blood, the tive control must be negative, and positive control readings patient’s renal function, the presence of a urinary tract infec- should agree with the published value. Results that do not tion, the patient’s dietary intake, and the age of the specimen agree with the published values must be resolved through the (Table 5–1). testing of additional strips and controls (see Chapter 1). Demonstration of chemically acceptable reagent strips Clinical Significance does not entirely rule out the possibility of inaccurate results. The importance of urinary pH is primarily as an aid in de- Interfering substances in the urine, technical carelessness, and termining the existence of systemic acid–base disorders of color blindness also produce errors. Reagent strip manufactur- metabolic or respiratory origin and in the management of ers have published information concerning the limitations urinary conditions that require the urine to be maintained (e.g., interfering substances, sensitivities) of their chemical re- at a specific pH. In respiratory or metabolic acidosis not re- actions, and laboratory personnel should be aware of these lated to renal function disorders, the urine is acidic; con- conditions. As mentioned in Chapter 4, a primary example of versely, if respiratory or metabolic alkalosis is present, the 3920_Ch05_071-098 23/01/14 10:14 AM Page 74 74 Part Two | Urinalysis SUMMARY 5-1 Reagent Strip Testing Table 5–1 Causes of Acid and Alkaline Urine Acid Urine Alkaline Urine Care of Reagent Strips 1. Store with desiccant in an opaque, tightly closed Emphysema Hyperventilation container. Diabetes mellitus Vomiting 2. Store below 30°C; do not freeze. Starvation Renal tubular acidosis 3. Do not expose to volatile fumes. Dehydration Presence of urease- 4. Do not use past the expiration date. producing bacteria 5. Do not use if chemical pads become discolored. Diarrhea Vegetarian diet 6. Remove strips immediately prior to use. Presence of acid-producing Old specimens Technique bacteria (Escherichia coli) 1. Mix specimen well. High-protein diet 2. Let refrigerated specimens warm to room tempera- Cranberry juice ture before testing. Medications (methenamine 3. Dip the strip completely, but briefly, into specimen. mandelate [Mandelamine], fosfomycin tromethamine 4. Remove excess urine by withdrawing the strip against [Monurol]) the rim of the container and by blotting the edge of the strip. 5. Compare reaction colors with the manufacturer’s pH discourages formation of the calculi. Knowledge of urinary chart under a good light source at the specified time. pH is important in the identification of crystals observed dur- 6. Perform backup tests when indicated. ing microscopic examination of the urine sediment. This will 7. Be alert for the presence of interfering substances. be discussed in detail in Chapter 6. Maintaining an acidic urine can be valuable in treating 8. Understand the principles and significance of the test; urinary tract infections caused by urea-splitting organisms read package inserts. because they do not multiply as readily in an acidic medium. 9. Relate chemical findings to each other and to the These same organisms are also responsible for the highly physical and microscopic urinalysis results. alkaline pH found in specimens that have been allowed to sit Quality Control unpreserved for extended periods. Urinary pH is controlled 1. Test open bottles of reagent strips with known posi- primarily by dietary regulation, although medications also tive and negative controls every 24 hours. may be used. Persons on high-protein and high-meat diets tend to produce acidic urine, whereas urine from vegetarians 2. Resolve control results that are out of range by further is more alkaline, due to the formation of bicarbonate following testing. digestion of many fruits and vegetables. An exception to the 3. Test reagents used in backup tests with positive and rule is cranberry juice, which produces an acidic urine and negative controls. has long been used as a home remedy for minor bladder in- 4. Perform positive and negative controls on new fections because it inhibits the colonization of certain urinary reagents and newly opened bottles of reagent strips. pathogens. People who are prone to frequent urinary tract in- 5. Record all control results and reagent lot numbers. fections are often advised to drink cranberry juice or take over-the-counter cranberry pills. Medications prescribed for urinary tract infections, such as methenamine mandelate (Mandelamine) and fosfomycin tromethamine (Monurol) are urine is alkaline. Therefore, a urinary pH that does not con- metabolized to produce an acidic urine. form to this pattern may be used to rule out the suspected The pH of freshly excreted urine does not reach above 8.5 condition, or, as discussed in Chapter 3, it may indicate a in normal or abnormal conditions. A pH above 8.5 is associ- disorder resulting from the kidneys’ inability to secrete or to ated with an improperly preserved specimen and indicates that reabsorb acid or base. a fresh specimen should be obtained to ensure the validity of The precipitation of inorganic chemicals dissolved in the the analysis. urine forms urinary crystals and renal calculi. This precipita- tion depends on urinary pH and can be controlled by main- taining the urine at a pH that is incompatible with the TECHNICAL TIP Collecting specimens in containers precipitation of the particular chemicals causing the calculi other than the single-use laboratory-supplied containers formation. For example, calcium oxalate, a frequent con- can produce a pH above 8.5 if alkaline detergent remains stituent of renal calculi, precipitates primarily in acidic and in the container. not alkaline urine. Therefore, maintaining urine at an alkaline 3920_Ch05_071-098 23/01/14 10:14 AM Page 75 Chapter 5 | Chemical Examination of Urine 75 SUMMARY 5-2 Clinical Significance of Protein Urine pH Of the routine chemical tests performed on urine, the most in- Respiratory or metabolic acidosis/ketosis dicative of renal disease is the protein determination. Proteinuria is often associated with early renal disease, making the urinary Respiratory or metabolic alkalosis protein test an important part of any physical examination. Nor- Defects in renal tubular secretion and reabsorption of mal urine contains very little protein: usually, less than 10 mg/dL acids and bases—renal tubular acidosis or 100 mg per 24 hours is excreted. This protein consists pri- Renal calculi formation and prevention marily of low-molecular-weight serum proteins that have been Treatment of urinary tract infections filtered by the glomerulus and proteins produced in the geni- tourinary tract. Due to its low molecular weight, albumin is the Precipitation/identification of crystals major serum protein found in normal urine. Even though it is Determination of unsatisfactory specimens present in high concentrations in the plasma, the normal uri- nary albumin content is low because the majority of albumin presented to the glomerulus is not filtered, and much of the Reagent Strip Reactions filtered albumin is reabsorbed by the tubules. Other proteins include small amounts of serum and tubular microglobulins; The Multistix and Chemstrip brands of reagent strips measure Tamm-Horsfall protein (uromodulin) produced by the renal urine pH in 0.5- or 1-unit increments between pH 5 and 9. To tubular epithelial cells; and proteins from prostatic, seminal, differentiate pH units throughout this wide range, both man- and vaginal secretions. (Uromodulin is a more recent name for ufacturers use a double-indicator system of methyl red and Tamm-Horsfall protein. Uromodulin is routinely produced in bromthymol blue. Methyl red produces a color change from the distal convoluted tubule. As will be discussed in Chapter 6, red to yellow in the pH range 4 to 6, and bromthymol blue uromodulin forms the matrix of casts formed in the distal con- turns from yellow to blue in the range of 6 to 9. Therefore, in voluted tubule.) the pH range 5 to 9 measured by the reagent strips, one sees colors progressing from orange at pH 5 through yellow and Clinical Significance green to a final deep blue at pH 9. Demonstration of proteinuria in a routine analysis does not Methyl red + H+ → bromthymol blue – H+ always signify renal disease; however, its presence does require (Red-orange → yellow) (green → blue) additional testing to determine whether the protein represents a normal or a pathologic condition. Clinical proteinuria is in- No known substances interfere with urinary pH measure- dicated at 30 mg/dL or greater (300 mg/L).7 The causes of pro- ments performed by reagent strips. teinuria are varied and can be grouped into three major categories: prerenal, renal, and postrenal, based on the origin of the protein. TECHNICAL TIP Care must be taken to prevent run-over Prerenal Proteinuria between the pH testing area and the adjacent, highly acidic protein testing area on Multistix, as this may pro- As the name implies, prerenal proteinuria is caused by condi- duce a falsely acidic reading in an alkaline urine. tions affecting the plasma prior to its reaching the kidney and, therefore, is not indicative of actual renal disease. This condition is frequently transient, caused by increased levels of low-molecular-weight plasma proteins such as hemoglobin, SUMMARY 5-3 pH Reagent Strip myoglobin, and the acute phase reactants associated with infection and inflammation. The increased filtration of these Reagents Methyl red, bromthymol blue proteins exceeds the normal reabsorptive capacity of the renal tubules, resulting in an overflow of the proteins into the urine. Sensitivity Multistix: 5.0 to 8.5 in 0.5 increments Because reagent strips detect primarily albumin, prerenal pro- Chemstrip: 5.0 to 9.0 in 1.0 increments teinuria is usually not discovered in a routine urinalysis. Sources of error/ No known interfering substances interference: Bence Jones Protein Run-over from adjacent pads A primary example of proteinuria due to increased serum pro- Old specimens tein levels is the excretion of Bence Jones protein by persons with multiple myeloma. In multiple myeloma, a proliferative Correlations with Nitrite disorder of the immunoglobulin-producing plasma cells, the other tests: serum contains markedly elevated levels of monoclonal im- Leukocytes munoglobulin light chains (Bence Jones protein). This low- Microscopic molecular-weight protein is filtered in quantities exceeding the tubular reabsorption capacity and is excreted in the urine. 3920_Ch05_071-098 23/01/14 10:14 AM Page 76 76 Part Two | Urinalysis Suspected cases of multiple myeloma must be diagnosed by occurrence in persons with both type 1 and type 2 diabetes performing serum electrophoresis and immunoelectrophore- mellitus. Onset of renal complications can first be predicted sis. The screening test for Bence Jones protein is not routinely by detection of microalbuminuria, and the progression of performed, as cases of multiple myeloma are easily detected renal disease can be prevented through better stabilization by chemical methods (see the Historical Note, Screening Test of blood glucose levels and control of hypertension. The pres- for Bence Jones Protein). ence of microalbuminuria is also associated with an increased risk of cardiovascular disease. 8, 9 Renal Proteinuria Orthostatic (Postural) Proteinuria Proteinuria associated with true renal disease may be the result of either glomerular or tubular damage. A persistent benign proteinuria occurs frequently in young adults and is termed orthostatic proteinuria, or postural Glomerular Proteinuria proteinuria. It occurs following periods spent in a vertical pos- When the glomerular membrane is damaged, selective filtration ture and disappears when a horizontal position is assumed. is impaired, and increased amounts of serum protein and even- Increased pressure on the renal vein when in the vertical tually red and white blood cells pass through the membrane position is believed to account for this condition. Patients sus- and are excreted in the urine. Conditions that present the pected of orthostatic proteinuria are requested to empty the glomerular membrane with abnormal substances (e.g., amyloid bladder before going to bed, collect a specimen immediately material, toxic substances, and the immune complexes found upon arising in the morning, and collect a second specimen in lupus erythematosus and streptococcal glomerulonephritis) after remaining in a vertical position for several hours. Both are major causes of proteinuria due to glomerular damage. specimens are tested for protein, and if orthostatic proteinuria Increased pressure from the blood entering the glomerulus is present, a negative reading will be seen on the first morning may override the selective filtration of the glomerulus, causing specimen, and a positive result will be found on the second increased albumin to enter the filtrate. This condition may be specimen. reversible, such as occurs during strenuous exercise and dehy- Tubular Proteinuria dration or is associated with hypertension. Proteinuria that occurs during the latter months of pregnancy may indicate a Increased albumin is also present in disorders affecting tubu- pre-eclamptic state and should be considered by the physician lar reabsorption because the normally filtered albumin can in conjunction with other clinical symptoms, such as hyper- no longer be reabsorbed. Other low-molecular-weight pro- tension, to determine if this condition exists. teins that are usually reabsorbed are also present. Causes of The discovery of protein, particularly in a random sample, tubular dysfunction include exposure to toxic substances and is not always of pathologic significance, because several benign heavy metals, severe viral infections, and Fanconi syndrome. causes of renal proteinuria exist. Benign proteinuria is usually The amount of protein that appears in the urine following transient and can be produced by conditions such as strenuous glomerular damage ranges from slightly above normal to exercise, high fever, dehydration, and exposure to cold. 4 g/day, whereas markedly elevated protein levels are seldom seen in tubular disorders. Microalbuminuria The development of diabetic nephropathy leading to reduced Postrenal Proteinuria glomerular filtration and eventual renal failure is a common Protein can be added to a urine specimen as it passes through the structures of the lower urinary tract (ureters, bladder, ure- thra, prostate, and vagina). Bacterial and fungal infections HISTORICAL NOTE Screening Test for Bence Jones HISTORICAL NOTE Protein Microalbuminuria Testing Unlike other proteins, which coagulate and remain coagu- lated when exposed to heat, Bence Jones protein coagulates Before the development of current reagent strip methods at temperatures between 40°C and 60°C and dissolves that are specific for albumin, detection of microalbumin- when the temperature reaches 100°C. Therefore, a speci- uria required collection of a 24-hour urine specimen. men that appears turbid between 40°C and 60°C and clear Specimens were tested using quantitative procedures for at 100°C can be suspected of containing Bence Jones pro- albumin. Results were reported in mg of albumin/24 hours tein. Interference due to other precipitated proteins can or as the albumin excretion (AER) in µg/min. With these be removed by filtering the specimen at 100°C and observ- methods, microalbumin was considered significant when ing the specimen for turbidity as it cools to between 40°C 30 to 300 mg of albumin is excreted in 24 hours or the and 60°C. AER is 20 to 200 µg/min. 3920_Ch05_071-098 23/01/14 10:14 AM Page 77 Chapter 5 | Chemical Examination of Urine 77 and inflammations produce exudates containing protein from can be difficult. Reporting of trace values may be a laboratory the interstitial fluid. The presence of blood as the result of option. injury or menstrual contamination contributes protein, as does the presence of prostatic fluid and large amounts of pH 3.0 spermatozoa. Indicator + protein protein + H+ (Yellow) indicator – H+ Reagent Strip Reactions (blue-green) Traditional reagent strip testing for protein uses the principle Reaction Interference of the protein error of indicators to produce a visible colori- metric reaction. Contrary to the general belief that indicators The major source of error with reagent strips occurs with highly produce specific colors in response to particular pH levels, buffered alkaline urine that overrides the acid buffer system, pro- certain indicators change color in the presence of protein even ducing a rise in pH and a color change unrelated to protein con- though the pH of the medium remains constant. This is be- centration. Likewise, a technical error of allowing the reagent cause protein (primarily albumin) accepts hydrogen ions from pad to remain in contact with the urine for a prolonged period the indicator. The test is more sensitive to albumin because may remove the buffer. False-positive readings are obtained albumin contains more amino groups to accept the hydrogen when the reaction does not take place under acidic conditions. ions than other proteins. Depending on the manufacturer, the Highly pigmented urine and contamination of the container with protein area of the strip contains either tetrabromophenol blue quaternary ammonium compounds, detergents, and antiseptics (Multistix) or 3',3",5',5"-tetrachlorophenol, 3,4,5,6-tetrabro- also cause false-positive readings. A false-positive trace reading mosulfonphthalein (Chemstrip), and an acid buffer to main- may occur in specimens with a high specific gravity. tain the pH at a constant level. At a pH level of 3, both indicators appear yellow in the absence of protein; however, Sulfosalicylic Acid Precipitation Test as the protein concentration increases, the color progresses The sulfosalicylic acid (SSA) test is a cold precipitation test through various shades of green and finally to blue. Readings that reacts equally with all forms of protein. Various concen- are reported in terms of negative, trace, 1+, 2+, 3+, and 4+; trations and amounts of SSA can be used to precipitate protein, or the semiquantitative values of 30, 100, 300, or 2000 mg/dL and methods vary greatly among laboratories. All precipitation corresponding to each color change. Trace values are consid- tests must be performed on centrifuged specimens to remove ered to be less than 30 mg/dL. Interpretation of trace readings any extraneous contamination. Based on the protocol of the laboratory, an SSA test may be performed in certain situations. The procedure is included in this section to serve as a reference if needed (Procedure 5–2).5 SUMMARY 5-4 Clinical Significance of Urine Protein Testing for Microalbuminuria Prerenal Tubular Disorders Several semiquantitative reagent strip methods have been Intravascular hemolysis Fanconi syndrome developed so that patients at risk for renal disease can be monitored using random or first morning specimens. These Muscle injury Toxic agents/heavy metals methods are based on immunochemical assays for albumin or Acute phase reactants Severe viral infections albumin-specific reagent strips that also measure creatinine to Multiple myeloma produce an albumin:creatinine ratio. Immunochemical assays include the Micral-Test (Roche Renal Postrenal Diagnostics, Indianapolis, IN) and the ImmunoDip (Sakisui Glomerular disorders Lower urinary tract infections/ Diagnostics, Framingham, MA). Both reagent strips are read inflammation visually, and first morning specimens are recommended. Immune complex Injury/trauma Micral-Test reagent strips contain a gold-labeled antihu- disorders man albumin antibody-enzyme conjugate. Strips are dipped Amyloidosis Menstrual contamination into the urine up to a level marked on the strip and held for 5 seconds. Albumin in the urine binds to the antibody. The Toxic agents Prostatic fluid/spermatozoa bound and unbound conjugates move up the strip by wicking Diabetic nephropathy Vaginal secretions Strenuous exercise Dehydration TECHNICAL TIP The specific gravity of the urine speci- Hypertension men should be considered in evaluating urine protein be- Pre-eclampsia cause a trace protein in a dilute specimen is more Orthostatic or postural proteinuria significant than in a concentrated specimen. 3920_Ch05_071-098 23/01/14 10:14 AM Page 78 78 Part Two | Urinalysis SUMMARY 5-5 Protein Reagent Strip PROCEDURE 5-2 Reagents Multistix: Tetrabromophenol blue Sulfosalicylic Acid Precipitation Test Chemstrip: 3’,3’’,5’,5’’-tetrachlorophenol 1. Add 3 mL of 3% SSA reagent to 3 mL of centrifuged 3,4,5,6-tetrabromosulfophthalein urine. Sensitivity Multistix: 15 to 30 mg/dL albumin 2. Mix by inversion and observe for cloudiness. Chemstrip: 6 mg/dL albumin 3. Grade the degree of turbidity (see table, following). Sources of error/interference False-positive: Table Reporting SSA Turbidity Highly buffered interference alkaline urine Grade Turbidity Protein Range Pigmented specimens, phenazopyridine (mg/dL) Negative No increase in Less than 6 Quaternary ammonium compounds turbidity (detergents) Trace Noticeable turbidity 6–30 Antiseptics, chlorhexidine 1+ Distinct turbidity, 30–100 Loss of buffer from prolonged exposure no granulation of the strip to the specimen reagent 2+ Turbidity, granulation, 100–200 High specific gravity no flocculation False-negative 3+ Turbidity, granulation, 200–400 Proteins other than albumin flocculation Microalbuminuria 4+ Clumps of protein Greater than Correlations Blood 400 with other tests: Nitrite Leukocytes particles. The color intensity of the bands is compared against Microscopic the manufacturer’s color chart. A darker bottom band repre- sents less 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. A darker bottom band is negative, equal band color is borderline, and a darker top band represents action. Unbound conjugates are removed in a captive zone by positive results. combining with albumin embedded in the strip. The urine albumin–bound conjugates continue up the strip and reach an Albumin:Creatinine Ratio area containing enzyme substrate. The conjugated enzyme re- acts with the substrate, producing colors ranging from white The Clinitek Microalbumin reagent strips and the Multistix Pro to red. The amount of color produced represents the amount reagent strips (Siemens Healthcare Diagnostics, Deerfield, IN) of albumin present in the urine. The color is compared with a provide simultaneous measurement of albumin/protein and chart on the reagent strip bottle after 1 minute. Results range creatinine that permits an estimation of the 24-hour microal- from 0 to 10 mg/dL. bumin excretion.10 As discussed in Chapter 3, creatinine is The ImmunoDip reagent strip uses an immunochromo- produced and excreted at a consistent rate for each individual. graphic technique. Strips are individually packaged in specially Therefore, by comparing the albumin excretion to the creati- designed containers. The container is placed in the urine spec- nine excretion, the albumin reading can be corrected for over- imen for 3 minutes. A controlled amount of urine enters the hydration and dehydration in a random sample. In addition to container through a vent hole. The urine encounters blue latex including creatinine on the reagent strip, the albumin low-test particles coated with antihuman albumin antibody. Albumin pad is changed to a dye-binding reaction that is more specific in the urine binds with the coated particles. The bound and for albumin than the protein error of indicators’ reaction on unbound particles continue to migrate up the strip. The mi- strips measuring protein. gration is controlled by the size of the particles; unbound par- Reagent Strip Reactions ticles do not migrate as far as the bound particles. First a blue band is formed by the unbound particles. The bound particles Albumin continue to migrate and form a second blue band further up Albumin reagent strips use the dye bis(3',3"-diiodo-4', the strip. The top band therefore represents the bound particles 4"-dihydroxy-5',5"-dinitrophenyl)-3,4,5,6-tetrabromo (urine albumin) and the bottom band represents unbound sulphonphthalein (DIDNTB), which has a higher sensitivity 3920_Ch05_071-098 23/01/14 10:14 AM Page 79 Chapter 5 | Chemical Examination of Urine 79 and specificity for albumin. Whereas conventional protein along with pads for glucose, ketones, blood, nitrite, leukocyte reagent pads have a sensitivity of 30 mg/dL or greater and may esterase, pH, bilirubin, and specific gravity. Urobilinogen is not include proteins other than albumin, the DIDNTB strips can included on these strips. The strips can be read manually or on measure albumin between 8 and 15 mg/dL (80 to 150 mg/L) automated Clinitek instruments. The protein-high reaction uses without inclusion of other proteins. Reaction interference by the protein error of indicators principle and the protein-low highly buffered alkaline urine (always a concern with conven- reaction is the previously discussed dye-binding method. tional reagent strips) is controlled by using paper treated with Results are reported as the protein:creatinine ratio, although the bis-(heptapropylene glycol) carbonate. Addition of polymethyl protein-low result is used in the calculation. Results from the vinyl ether decreases the nonspecific binding of polyamino Clinitek are automatically calculated. Results are reported as acids to the albumin pad. Colors range from pale green to aqua normal or abnormal. A result of normal dilute indicates that the blue. Falsely elevated results can be caused by visibly bloody specimen should be recollected, making sure it is a first morn- urine, and abnormally colored urines may interfere with the ing specimen. readings.2 When the reagent strip is read manually, a manufacturer- supplied chart is used to determine the ratio based on the results Creatinine of the protein-high, protein-low, and creatinine readings. When The principle of the reagent strip for creatinine is based on the using this chart, the higher of the protein-low or protein-high pseudoperoxidase activity of copper-creatinine complexes. The result is used2 (Fig. 5–1). reaction follows the same principle as the reaction for blood on the reagent strips discussed later in this chapter. Reagent strips contain copper sulfate (CuSO4), 3,3',5,5'-tetramethyl- Glucose benzidine (TMB), and diisopropyl benzene dihydroperoxide Because of its value in the detection and monitoring of diabetes (DBDH). Creatinine in the urine combines with the copper mellitus, the glucose test is the most frequently performed sulfate to form copper-creatinine peroxidase. This reacts with chemical analysis on urine. Due to the nonspecific symptoms the peroxide DBDH, releasing oxygen ions that oxidize the associated with the onset of diabetes, it is estimated that more chromogen TMB and producing a color change from orange than half of the cases in the world are undiagnosed. Therefore, through green to blue.7 blood and urine glucose tests are included in all physical ex- aminations and are often the focus of mass health screening CuSO4 + CRE → Cu(CRE) peroxidase programs. Early diagnosis of diabetes mellitus through blood Cu(CRE) peroxidase and urine glucose tests provides a greatly improved prognosis. DBDH + TMB oxidized TMB + H2O Using currently available reagent strip methods for both blood (peroxidase) (chromogen) (orange to blue) and urine glucose testing, patients can monitor themselves at Results are reported as 10, 50, 100, 200, 300 mg/dL, or home and can detect regulatory problems prior to the devel- 0.9, 4.4, 8.8, 17.7, or 26.5 mmol/L of creatinine. opment of serious complications. Reagent strips are unable to detect the absence of creati- nine. Falsely elevated results can be caused by visibly bloody Clinical Significance urine and the presence of the gastric acid–reducing medication Under normal circumstances, almost all the glucose filtered by cimetidine (Tagamet). Abnormally colored urines also may in- the glomerulus is actively reabsorbed in the proximal convo- terfere with the readings. luted tubule; therefore, urine contains only minute amounts No creatinine readings are considered abnormal, as creati- of glucose. Tubular reabsorption of glucose is by active trans- nine is normally present in concentrations of 10 to 300 mg/dL. port in response to the body’s need to maintain an adequate The purpose of the creatinine measurement is to correlate the concentration of glucose. Should the blood level of glucose be- albumin concentration to the urine concentration, producing a come elevated (hyperglycemia), as occurs in diabetes mellitus, semiquantitative albumin:creatinine ratio (A:C) ratio. Reported Albumin/Protein:Creatinine Ratio Protein Creatinine Result (mg/dL) Automated and manual methods are available for determining Result (mg/dL) 10 50 100 200 300 the A:C ratio based on the previously discussed reactions. The Clinitek Microalbumin reagent strips are designed for instru- Negative Recollect* No rm mental use only. Strips are read on Clinitek Urine Chemistry 15 al Ab Analyzers. The strips measure only albumin and creatinine and no calculate the A:C ratio. Results are displayed and printed out 30 rm 100, 300, al for albumin, creatinine, and the A:C ratio in both conventional or 2000 and S.I. units. Abnormal results for the A:C ratio are 30 to *Specimen is too dilute to determine ratio result accurately. Repeat test on new 300 mg/g or 3.4 to 33.9 mg/mmol.10 specimen, preferably a first-morning collection. The Siemens Multistix Pro 10 reagent strips include reagent Figure 5–1 A protein:creatinine ratio determination chart. (Image pads for creatinine, protein-high and protein-low (albumin), adapted from Bayer HealthCare LLC, Elkhart, IN.) 3920_Ch05_071-098 23/01/14 10:14 AM Page 80 80 Part Two | Urinalysis Hyperglycemia that occurs during pregnancy and disap- SUMMARY 5-6 Microalbumin Testing pears after delivery is called gestational diabetes. The onset of Immunologic Tests the hyperglycemia and glycosuria is normally around the sixth month of pregnancy, although glycosuria may occur sooner. Micral-Test Hormones secreted by the placenta block the action of insulin, Principle: Enzyme immunoassay resulting in insulin resistance and hyperglycemia. Detection of Sensitivity: 0 to 10 mg/dL gestational diabetes is important to the welfare of the baby, be- Reagents: Gold-labeled antibody cause glucose crosses the placenta whereas insulin does not. B-galactosidase The baby develops high glucose levels, causing the baby’s pan- creas to produce more insulin. The excess glucose presented Chlorophenol red galactoside to the baby is stored as fat, resulting in a large baby (macroso- Interference: False-negative: Dilute urine mia) at risk for obesity and later type 2 diabetes. Women who ImmunoDip have gestational diabetes also are prone to developing type 2 Principle: Immunochromographics diabetes mellitus in later years. Hyperglycemia of nondiabetic origin is seen in a variety of Sensitivity: 1.2 to 8.0 mg/dL disorders and also produces glycosuria. Many of these disorders Reagents: Antibody-coated blue latex particles are associated with hormonal function and include pancreatitis, Interference: False-negative: Dilute urine acromegaly, Cushing syndrome, hyperthyroidism, pheochromo- Albumin:Creatinine Ratio cytoma, and thyrotoxicosis. The hormones glucagon, epinephrine, cortisol, thyroxine, and growth hormone, which are increased in Clinitest Microalbumin Strips/Multistix-Pro these disorders, work in opposition to insulin, thereby producing Principle: Sensitive albumin tests related to creatinine hyperglycemia and glucosuria. Whereas a primary function of in- concentration to correct for patient hydration sulin is to convert glucose to glycogen for storage (glycogenesis), Reagents: these opposing hormones cause the breakdown of glycogen to Albumin: dye bis(3’,3’’-diiodo-4’,4’’-dihydroxy-5’,5’’- glucose (glycogenolysis), resulting in increased levels of circulat- dinitrophenyl)-3,4,5,6-tetrabromo sulphonphtalein ing glucose. Epinephrine is also a strong inhibitor of insulin se- (DIDNTB) cretion and is increased when the body is subjected to severe stress, which accounts for the glucosuria seen in conjunction with Creatinine: copper sulfate (CuSO4), 3,3’,5,5’-tetram- cerebrovascular trauma and myocardial infarction. ethylbenzidine (TMB), and diisopropyl benzene Glycosuria occurs in the absence of hyperglycemia when dihydroperoxide (DBDH) the reabsorption of glucose by the renal tubules is compro- Sensitivity: mised. This is frequently referred to as “renal glycosuria” and Albumin: 10 to 150 mg/L is seen in end-stage renal disease, cystinosis, and Fanconi syn- Creatinine: 10 to 300 mg/dL, 0.9 to 26.5 mmol/L drome. Glycosuria not associated with gestational diabetes is occasionally seen as a result of a temporary lowering of the Interference: renal threshold for glucose during pregnancy. Visibly bloody or abnormally colored urine Creatinine: Cimetidine: False positive SUMMARY 5-7 Clinical Significance of Urine Glucose the tubular transport of glucose has reached its renal threshold, and glucose appears in the urine. The blood level at which Hyperglycemia-Associated Renal-Associated tubular reabsorption stops (renal threshold) for glucose is Diabetes mellitus Fanconi syndrome approximately 160 to 180 mg/dL. Blood glucose levels fluctu- Pancreatitis Advanced renal ate, and a nonfasting normal person may have glycosuria Pancreatic cancer disease following a meal containing a high glucose content. Therefore, Osteomalacia Acromegaly the most informative glucose results are obtained from speci- Cushing syndrome Pregnancy mens collected under controlled conditions. Fasting prior to the collection of samples for screening tests is recommended. Hyperthyroidism For purposes of diabetes monitoring, specimens are usually Pheochromocytoma tested 2 hours after meals. A first morning specimen does not Central nervous system damage always represent a fasting specimen because glucose from an evening meal may remain in the bladder overnight, and pa- Stress tients should be advised to empty the bladder and collect the Gestational diabetes second specimen.2 3920_Ch05_071-098 23/01/14 10:14 AM Page 81 Chapter 5 | Chemical Examination of Urine 81 Reagent Strip (Glucose Oxidase) Reaction SUMMARY 5-8 Glucose Reagent Strip The glucose oxidase procedure provides a specific test for glu- Reagents Multistix cose. Reagent strips employ the glucose oxidase testing method by impregnating the testing area with a mixture of glucose ox- Glucose oxidase idase, peroxidase, chromogen, and buffer to produce a double Peroxidase sequential enzyme reaction. In the first step, glucose oxidase Potassium iodide catalyzes a reaction between glucose and room air (oxygen) Chemstrip to produce gluconic acid and peroxide. In the second step, peroxidase catalyzes the reaction between peroxide and chro- Glucose oxidase mogen to form an oxidized colored compound that is pro- Peroxidase duced in direct proportion to the concentration of glucose. Tetramethylbenzidine Sensitivity Multistix: 75 to 125 mg/dL Glucose oxidase 1. Glucose + O2 (air) gluconic acid + H2O2 Chemstrip: 40 mg/dL Peroxidase Interference False-positive 2. H2O2 + chromogen oxidized Contamination by oxidizing colored chromogen + H2O agents and detergents Reagent strip manufacturers use several different chro- False-negative mogens, including potassium iodide (green to brown) (Multistix) High levels of ascorbic acid and tetramethylbenzidine (yellow to green) (Chemstrip). Urine High levels of ketones glucose may be reported in terms of negative, trace, 1+, 2+, 3+, and 4+; however, the color charts also provide quantitative High specific gravity measurements ranging from 100 mg/dL to 2 g/dL, or 0.1% to Low temperatures 2%. The American Diabetes Association recommends quantita- Improperly preserved specimens tive reporting. Correlations with Ketones Reaction Interference other tests Protein Because the glucose oxidase method is specific for glucose, false-positive reactions are not obtained from other urinary con- stituents, including reducing sugars that may be present. False- copper sulfate to cuprous oxide in the presence of alkali and positive reactions may occur, however, if containers become heat. A color change progressing from a negative blue (CuSO4) contaminated with peroxide or strong oxidizing detergents. through green, yellow, and orange/red (Cu2O) occurs when the Substances that interfere with the enzymatic reaction or reaction takes place. strong reducing agents, such as ascorbic acid, that prevent ox- idation of the chromogen may produce false-negative results. Heat CuSO4 (cupric sulfide) + reducing substance To minimize interference from ascorbic acid, reagent strip man- Alkali ufacturers are incorporating additional chemicals into the test Cu2O (cuprous oxide) + oxidized substance → color pads. An example is iodate that oxidizes ascorbic acid so that it (blue/green → orange/red) cannot interfere with the oxidation of the chromogen. Product literature should be carefully reviewed for current information The classic Benedict solution was developed in 1908 and regarding all interfering substances. High levels of ketones also contained copper sulfate, sodium carbonate, and sodium cit- affect glucose oxidase tests at low glucose concentrations; how- rate buffer.11 Urine was added to the solution, heat was ap- ever, because high levels of ketones are usually accompanied plied, and the resulting precipitate was observed for color. A by marked glycosuria, this seldom presents a problem. High more convenient method that employs Benedict’s principle is specific gravity and low temperature may decrease the sensitiv- the Clinitest tablet (Siemens Healthcare Diagnostics, Deerfield, ity of the test. By far the greatest source of false-negative glucose IN). The tablets contain copper sulfate, sodium carbonate, results is the technical error of allowing specimens to remain sodium citrate, and sodium hydroxide. Upon addition of unpreserved at room temperature for extended periods, sub- the tablet to water and urine, heat is produced by the hydrol- jecting the glucose to bacterial degradation. ysis of sodium hydroxide and its reaction with sodium citrate, and carbon dioxide is released from the sodium carbonate Copper Reduction Test (Clinitest) to prevent room air from interfering with the reduction Measurement of glucose by the copper reduction method was reaction. Thick-walled tubes should be placed in a heat- one of the earliest chemical tests performed on urine. The test resistant rack and not held in the hand because the reaction relies on the ability of glucose and other substances to reduce heat could cause a burn. At the conclusion of the effervescent 3920_Ch05_071-098 23/01/14 10:14 AM Page 82 82 Part Two | Urinalysis reaction, the tube is gently shaken, and the color ranging from prevents breakdown of ingested galactose and results in failure blue to orange/red can be compared with the manufacturer’s to thrive and other complications, including death. All states color chart to determine the approximate amount of reducing have incorporated screening for galactosemia into their re- substance. quired newborn screening programs (see Chapter 8) because Care must be taken to observe the reaction closely as it is early detection followed by dietary restriction can control the taking place, because at high glucose levels, a phenomenon condition. Depending on the laboratory population Clinitest known as “pass through” may occur. When this happens, the is often performed on pediatric specimens from patients up to color produced passes through the orange/red stage and re- at least the age of 2 years. The appearance of other reducing turns to a green-brown color, and if not observed, a high glu- sugars is usually of minimal clinical significance, and lactose cose level may be reported as negative. An alternate method is frequently found in the urine of nursing mothers. using two drops instead of five drops of urine can minimize the occurrence of “pass through.” A separate color chart must be used to interpret the reaction. This chart provides values up Ketones to 5 g/dL, whereas the five-drop method is limited to 2 g/dL. The term “ketones” represents three intermediate products of The sensitivity of Clinitest to glucose is reduced to a min- fat metabolism, namely, acetone (2%), acetoacetic acid (20%), imum of 200 mg/dL so the Clinitest cannot be used as a con- and β -hydroxybutyrate (78%). Normally, measurable amounts firmatory test for glucose. As a nonspecific test for reducing of ketones do not appear in the urine, because all the metabo- substances, Clinitest is subject to interference from other lized fat is completely broken down into carbon dioxide and reducing sugars, including galactose, lactose, fructose, maltose, water. However, when the use of available carbohydrate as the pentoses, ascorbic acid, certain drug metabolites, and antibi- major source of energy becomes compromised, body stores otics such as the cephalosporins. Therefore, Clinitest does not of fat must be metabolized to supply energy. Ketones are then provide a confirmatory test for glucose. detected in urine. Clinitest tablets are very hygroscopic and should be stored in their tightly closed packages. A strong blue color in the un- Clinical Significance used tablets suggests deterioration due to moisture accumula- tion, as does vigorous tablet fizzing. Clinical reasons for increased fat metabolism include the in- ability to metabolize carbohydrate, as occurs in diabetes mel- Clinical Significance of Clinitest litus; increased loss of carbohydrate from vomiting; and inadequate intake of carbohydrate associated with starvation In addition to glucose, commonly found reducing sugars in- and malabsorption. clude galactose, fructose, pentose, and lactose, of which galac- Testing for urinary ketones is most valuable in the man- tose is the most clinically significant. Galactose in the urine of agement and monitoring of insulin-dependent (type 1) diabetes a newborn represents an “inborn error of metabolism” in which mellitus. Ketonuria shows a deficiency in insulin, indicating lack of the enzyme galactose-1-phosphate uridyl transferase the need to regulate dosage. It is often an early indicator of in- sufficient insulin dosage in type 1 diabetes and in patients with diabetes who experience medical problems in addition to dia- PROCEDURE 5-3 betes. Increased accumulation of ketones in the blood leads to electrolyte imbalance, dehydration, and, if not corrected, aci- Clinitest Procedure dosis and eventual diabetic coma. 1. Place a thick glass test tube in a rack; add 5 drops of The use of multiple-test strips in hospital laboratories urine. often produces positive ketone tests unrelated to diabetes be- 2. Add 10 drops of distilled water to the urine in the cause the patient’s illness either prevents adequate intake or test tube. absorption of carbohydrates or produces an accelerated loss, 3. Drop one Clinitest tablet into the test tube and observe as in the case of vomiting. Weight-loss and eating disorder clin- the reaction until completion (cessation of boiling). ics can use a practical application of ketonuria produced by avoidance of carbohydrates to monitor patients. Frequent CAUTION: The reaction mixture gets very hot. Do strenuous exercise can cause overuse of available carbohydrates not touch the bottom area of the test tube. Use thick and produce ketonuria. glass test tube only. 4. Wait 15 seconds after boiling has stopped and gently shake the contents of the tube. 5. Compare the color of the mixture to the Clinitest TECHNICAL TIP Keep in mind that table sugar is sucrose, color chart and record the result in mg/dL or percent. a nonreducing sugar, and does not react with Clinitest or Observe for the possibility of the “pass-through” glucose oxidase strips and therefore cannot be used as a phenomenon. If present, repeat the procedure using control or in preparation of a laboratory exercise for glu- 2 drops of urine instead of 5 drops. cose testing.