Routine Urinalysis—the Chemical Examination

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Routine Urinalysis—the Chemical Examination


Learning Objectives


After studying this chapter, the student should be able to:




  1. 1. State the proper care and storage of commercial reagent strip and tablet tests and cite at least three potential causes of their deterioration.
  2. 2. Describe quality control procedures for commercial reagent strip and tablet tests.
  3. 3. Discuss appropriate specimen and testing techniques used with commercial reagent strip and tablet tests.
  4. 4. State the chemical principle used on reagent strips for measurement of the following:

  5. 5. Summarize the clinical significance of the following substances when present in urine and describe the chemical principles used on reagent strips to measure them:

  6. 6. Compare and contrast the sensitivity, specificity, and potential interferences of each commercial reagent strip and tablet test.
  7. 7. Compare and contrast the mechanisms for and the clinical significance of the following types of proteinurias:

  8. 8. Discuss the clinical features of the nephrotic syndrome and Fanconi’s syndrome, including the specific renal dysfunctions involved.
  9. 9. Compare and contrast the chemical principle, sensitivity, and specificity of the following tests for the detection of proteins in the urine:

  10. 10. Differentiate between hematuria and hemoglobinuria.
  11. 11. Discuss the clinical significance of myoglobin. Compare and contrast myoglobinuria and hemoglobinuria.
  12. 12. Discuss the limitations of leukocyte esterase and nitrite reagent strip tests for the detection of leukocyturia and bacteriuria.
  13. 13. Describe two physiologic mechanisms that result in glucosuria.
  14. 14. Compare and contrast the glucose reagent strip test and the copper reduction test for the measurements of sugars in urine.
  15. 15. Describe three conditions that result in ketonuria.
  16. 16. Briefly explain the metabolic pathway that results in ketone formation, state the relative concentrations of the three ketones formed, and discuss the reagent strip and tablet tests used to detect them.
  17. 17. Summarize the formation of bilirubin and urobilinogen, discuss their clinical significance, and describe three physiologic mechanisms that result in altered bilirubin metabolism.
  18. 18. Describe two chemical principles used by reagent strip tests to detect urine urobilinogen and compare their sensitivity, specificity, and limitations.
  19. 19. Compare and contrast the principle, sensitivity, specificity, and limitations of the following methods for detection of bilirubin in urine:

  20. 20. State the importance of ascorbic acid detection in urine, and describe methods used to detect ascorbic acid.
  21. 21. Identify reagent strip tests that are affected adversely by ascorbic acid, and explain the mechanism of interference.
  22. 22. Describe the role of reflex testing in urinalysis and discuss the correlation between results obtained in the chemical examination and what they imply for the microscopic examination.

Key Terms1



Reagent Strips


Commercial reagent strips are routinely used for chemical analysis of urine. Reagent strips enable rapid screening of urine specimens for pH, protein, glucose, ketones, blood, bilirubin, urobilinogen, nitrite, and leukocyte esterase. In addition, specific gravity and ascorbic acid can be determined by reagent strip, depending on the brand of strip used. Four commonly used brands of commercial reagent strips are Multistix (Siemens Healthcare Diagnostics Inc., Deerfield, IL), Chemstrips (Roche Diagnostics, Indianapolis, IN), and Aution Sticks (Arkray Inc., Kyoto, Japan). Most commercial reagent strips are available with single or multiple test pads on a reagent strip, which allows flexibility in test selection and cost containment.


A reagent strip is an inert plastic strip onto which reagent-impregnated test pads are bonded (Fig. 6.1). Chemical reactions take place after the strip is wetted with urine. Each reaction results in a color change that can be assessed visually or mechanically. By comparing the color change observed with the color chart supplied by the strip manufacturer, qualitative results for each reaction are determined. See Appendix A for samples of these color charts and the reporting formats provided by manufacturers on reagent strip containers. Depending on the test, results are reported (1) in concentration (milligrams per deciliter); (2) as small, moderate, or large; (3) using the plus system (1+, 2+, 3+, 4+); or (4) as positive, negative, or normal. The specific gravity and the pH are exceptions; these results are estimated in their respective units. Manufacturers do not use the same reporting terminology. For example, Multistix strips report glucose values less than 100 mg/dL as negative, whereas Chemstrips report these glucose results as normal. These minor inconsistencies among products can be confusing. Therefore, laboratorians must be aware of the reporting format, the chemical principles involved, and the specificity and sensitivity of each test included on the reagent strips used in their laboratory.



The chemical principles used on reagent strips are basically the same, with some manufacturers differing only in the determination of urobilinogen. See Appendix B for a tabular summary and comparison of reagent strip reaction principles, sensitivity, and specificity, which are also discussed throughout this chapter.


Reagent strips are available with a single test pad (e.g., Albustix, a single-protein test pad) or with a variety of test pad combinations. These combinations vary from 2 to 10 testpads per reagent strip and enable health-care providers to selectively screen urine specimens for only those constituents that interest them (e.g., Chemstrip 2 LN and Multistix 2 have only two pads: leukocyte esterase and nitrite tests). Some manufacturers also include a pad to account for urine color when automated reagent strip readers (i.e., reflectance photometers) are used.


Because reducing agents such as ascorbic acid have the potential to adversely affect several reagent strip test results, it is important that these and other potential interferences are detected or eliminated. In this regard, Chemstrip reagent strips use an iodate overlay on the blood test pad to eliminate ascorbic acid interference. The presence of interferences must be known to enable alternative testing when possible or to appropriately modify the results to be reported. Common interferences encountered in the chemical examination of urine and the effects these interferences have on urinalysis results are discussed for each reagent strip test in this chapter.


Care and Storage


Reagent strips, which are sometimes called dipsticks, are examples of state-of-the-art technology. Before the development of the first dry chemical dipstick test for glucose in the 1950s, all chemical tests on urine were performed individually in test tubes. Reagent strips have significantly (1) reduced the time required for testing, (2) reduced costs (e.g., reagents, personnel), (3) enhanced test sensitivity and specificity, and (4) decreased the amount of urine required for testing.


To ensure the integrity of reagent strips, their proper storage is essential, and the manufacturer’s directions must be followed. Each manufacturer provides a comprehensive product insert that outlines the chemical principles, reagents, storage, use, sensitivity, specificity, and limitations of its reagent strips. All reagent strips must be protected from moisture, chemicals, heat, and light. Any strips showing evidence of deterioration, contamination, or improper storage should be discarded. Tight-fitting lids, along with desiccants or drying agents within the product container, help eliminate test pad deterioration due to moisture. Fumes from volatile chemicals, acids, and bases can adversely affect the test pads and should be avoided. All reagent strip containers protect the reagent strips from ultraviolet rays and sunlight; however, the containers themselves must be protected to prevent fading of the color chart located on the label of the container. Reagent strips should be stored in their original containers at temperatures below 30°C (86°F); they are stable until the expiration date indicated on the label. To ensure accurate test results, all reagent strips—whether from a newly opened container or from one that has been opened for several months—must be periodically tested using appropriate control materials.


Quality Control Testing


Quality control testing of reagent strips not only ensures that the reagent strips are functioning properly, but also confirms the acceptable performance and technique of the laboratorian using them. Multiconstituent controls at two distinct levels (e.g., negative and positive) for each reaction must be used to check the reactivity of reagent strips. New containers or lot numbers of reagent strips must be checked “at a frequency defined by the laboratory, related to workload, suggested by the manufacturer, and in conformity with any applicable regulations.”1


Commercial or laboratory-prepared materials can serve as acceptable negative controls. Similarly, positive controls can be purchased commercially or prepared by the laboratory. Because of the time and care involved in making a multiconstituent control material that tests each parameter on the reagent strip, most laboratories purchase control materials. When control materials are tested, acceptable test performance is defined by each laboratory. Regardless of the control material used, care must be taken to enwsure that analyte values are within the critical detection levels for each parameter. For example, a protein concentration of 1 g/dL would be inappropriate as a control material because it far exceeds the desired critical detection level of 10 to 15 mg/dL.


An additional quality check on chemical and microscopic examinations, as well as on the laboratorian, involves aliquoting a well-mixed urine specimen from the daily workload and having a different laboratory (interlaboratory) or a technologist on each shift (intralaboratory) analyze the specimen. Interlaboratory duplicate testing checks the entire urinalysis procedure and detects innocuous changes when manual urinalyses are performed, such as variations in the speed of centrifugation and in centrifuge brake usage. Intralaboratory duplicate testing can also be used to evaluate the technical competency of laboratorians.


Tablet and Chemical Tests


Care and Storage


Commercial tablet tests (e.g., Ictotest, Clinitest, Acetest [all from Siemens Healthcare Diagnostics Inc.]) must be handled and stored according to the inserts provided by the manufacturers. These products are susceptible to deterioration from exposure to light, heat, and moisture. Therefore they should be visually inspected before each use and discarded if any of the following changes have occurred: tablet discolored, contamination or spoilage evident, incorrect storage, or past the expiration date. Note that the stability of the reaction tablets can decrease after opening because of repeated exposure to atmospheric moisture. To ensure tablet integrity, an appropriate quality control program must be employed.


Chemical tests such as the sulfosalicylic acid (SSA) precipitation test, the Hoesch test, and the Watson-Schwartz test require appropriately made and tested reagents. When new reagents are prepared, they should be tested in parallel with current “in-use” reagents to ensure equivalent performance. Chemical tests must also be checked according to the laboratory’s quality control program to ensure the reliability and reproducibility of test results obtained.


Quality Control Testing


As with reagent strips, tablet or chemical tests performed in the urinalysis laboratory must have quality control materials run to ensure the integrity of the reagents and the technique used in testing. Some commercial controls for reagent strips can also be used to check the integrity of Clinitest, Ictotest, and Acetest tablets. In addition, lyophilized chemistry controls or laboratory-made control materials can be used. For example, a chemistry albumin standard at an appropriate concentration (approximately 30 to 100 mg/dL) serves as a satisfactory control for performance of the SSA protein precipitation test.


Positive and negative quality control materials must be analyzed according to the frequency established in the laboratory’s policy. New tablets and reagents should be checked before they are placed into use and periodically thereafter.


Chemical Testing Technique


Reagent Strips


Although reagent strips are easy to use, proper technique is imperative to ensure accurate results. Box 6.1 summarizes an appropriate manual reagent strip testing technique. The manufacturer’s instructions provided with the reagent strips and tablet tests must be followed to obtain accurate results. Note that instructions vary among different manufacturers and that failure to read reaction results at the correct time could cause the reporting of erroneous results. For example, the reactions on Multistix strips must be read at the specific time indicated, which varies from 30 seconds to 2 minutes. In contrast, all reactions on Chemstrip brand strips are stabilized and can be read at 2 minutes.



A fresh, well-mixed, uncentrifuged specimen is used for testing. If the specimen is maintained at room temperature, it must be tested within 2 hours after collection to avoid erroneous results caused by changes that can occur in unpreserved urine1 (see Chapter 2, Table 2.3). If the urine specimen has been refrigerated, it should be allowed to warm up to room temperature before testing with reagent strips to avoid erroneous results. The specimen can be tested in the original collection container or after an aliquot is poured into a labeled centrifuge tube. The reagent strip should be briefly dipped into the urine specimen, wetting all test pads. Excess urine should be drained from the strip by drawing the edge of the strip along the rim of the container or by placing the strip edge on an absorbent paper. Inadequate removal of excess urine from the strip can cause contamination of one test pad with the reagents from another, whereas prolonged dipping of the strip causes the chemicals to leach from the test pad into the urine. Both of these actions can produce erroneous test results.


When reagent strips are read, the time required before full color development varies with the test parameter. To obtain reproducible and reliable results, the timing instructions provided by the manufacturer must be followed. Timing intervals can differ among reagent strips from the same manufacturer and among different manufacturers of the same test. For example, when a Multistix strip is used, the ketone test pad is read at 40 seconds; however, when Ketostix strips are used, the test area is read at 15 seconds. Some reagent strips have the flexibility of reading all test pads, except leukocytes, at any time between 60 and 120 seconds (e.g., Chemstrips), whereas others require the exact timing of each test pad for semiquantitated results (e.g., Multistix strips).


Visual interpretation of color varies slightly among individuals; therefore reagent strips should be read in a well-lit area with the strip held close to the color chart. The strip must be properly oriented to the chart before results are determined. Because of similar color changes by several of the test pads, improper orientation of the strip to the color chart is a potential source of error. (See Appendix A, Reagent Strip Color Charts.) Note that color changes appearing only along the edge of a reaction pad or after 2 minutes are diagnostically insignificant and should be disregarded. When reagent strips are read by automated instruments, the timing intervals are set by the factory. The advantage of automated instruments in reading reagent strips is their consistency in timing and color interpretation regardless of room lighting or testing personnel. Some instruments, however, are unable to identify and compensate for urine that is highly pigmented owing to medications. This can lead to false-positive reagent strip test results because the true color reaction is masked by the pigment present. Laboratorians should identify highly pigmented urine specimens and manually test them using reagent strips or alternative methods. The sensitivity and specificity of three brands of commercial reagent strips are discussed throughout this chapter and are summarized in tabular form in Appendix B.


Tablet and Chemical Tests


With each tablet test, the manufacturer’s directions must be followed exactly to ensure reproducible and reliable results. All chemical tests, such as the SSA precipitation test for protein, must be performed according to established written laboratory procedures. The laboratorian should know the sensitivity, specificity, and potential interferences for each test. Chemical and tablet tests are generally performed (1) to confirm results already obtained by reagent strip testing, (2) as an alternative method for highly pigmented urine, (3) because they are more sensitive for the substance of interest than the reagent strip test (e.g., Ictotest tablets), or (4) because the specificity of the test differs from that of the reagent strip test (e.g., Clinitest, SSA protein test).


Chemical Tests


Specific Gravity


Despite being a physical characteristic of urine, specific gravity is often determined during the chemical examination using commercial reagent strips. Provided in Table 6.1 is a listing of specific gravity values and associated conditions. For in-depth discussion of the clinical significance and renal tubular functions reflected by specific gravity as well as osmolality measurements, see Chapter 4, subsections Specific Gravity and Assessment of Renal Concentrating Ability.



Table 6.1























Clinical Significance of Urine Specific Gravity Results
Specific Gravity Indication or Cause
1.000 Physiologically impossible—the same as pure water; suspect adulteration of urine specimen
1.001–1.009 Dilute urine; associated with increased water intake or water diuresis (e.g., diuretics, inadequate secretion/action of ADH)
1.010–1.025 Indicates average solute and water intake and excretion
1.025–1.035 (1.040 maximum) Concentrated urine; associated with dehydration, fluid restriction, profuse sweating, osmotic diuresis
>1.040 Physiologically impossible; indicates presence of iatrogenic substance (e.g., radiographic contrast media, mannitol)

ADH, Antidiuretic hormone, also known as arginine vasopressin (AVP).


Methods available for measuring specific gravity differ in their ability to detect and measure solutes. Therefore it is important that health-care providers are informed of the test method used—its principle, sensitivity, specificity, and limitations. See Chapter 5 and Table 5.6 for a detailed discussion and comparison of the specific gravity methods: refractometry and reagent strip method.


Principle


The reagent strip specific gravity test does not measure the total solute content but only those solutes that are ionic. Keep in mind that only ionic solutes are involved in the renal concentrating and secreting ability of the kidneys and therefore this test has diagnostic value (see Chapter 4). Table 6.2 provides a summary of the specific gravity reaction principle, sensitivity, and specificity using selected reagent strip brands.



pH


Clinical Significance


The kidneys play a major role in regulating the acid–base balance of the body, as discussed in Chapter 3. The renal system, the pulmonary system, and blood buffers provide the means for maintaining homeostasis at a pH compatible with life. Normal daily metabolism generates endogenous acids and bases; in response, the kidneys selectively excrete acid or alkali. Normally, the urine pH varies from 4.5 to 8.0. The average individual excretes slightly acidic urine of pH 5.0 to 6.0 because endogenous acid production predominates. However, during and after a meal the urine produced is less acidic. This observation is known as the alkaline tide.


Urine pH can affect the stability of formed elements in urine. An alkaline pH enhances lysis of cells and degradation of the matrix of casts. Because pH values greater than 8.0 and less than 4.5 are physiologically impossible, they require investigation when obtained. The three most common reasons for a urine pH greater than 8.0 are (1) a urine specimen that was improperly preserved and stored, resulting in the proliferation of urease-producing bacteria and resultant increased pH; (2) an adulterated specimen (i.e., an alkaline agent was added to the urine after collection); and (3) the patient was given a highly alkaline substance (e.g., medication, therapeutic agent) that was subsequently excreted by the kidneys. In the latter situation, efforts should be made to ensure adequate hydration of the patient to prevent in vivo precipitation of normal urine solutes (e.g., ammonium biurate crystals), which can cause renal tubular damage.


Because the kidneys constantly maintain the acid–base balance of the body, ingestion of acids or alkali or any condition that produces acids or alkali directly affects the urine pH. Table 6.3 lists urine pH values and common causes associated with them. This ability of the kidneys to manipulate urine pH has many applications. An acid urine prevents stone formation by alkaline-precipitating solutes (e.g., calcium carbonate, calcium phosphate) and inhibits the development of UTI. An alkaline urine prevents the precipitation of and enhances the excretion of various drugs (e.g., sulfonamides, streptomycin, salicylate) and prevents stone formation from calcium oxalate, uric acid, and cystine crystals.



The urine pH provides valuable information for assessing and managing disease and for determining the suitability of a specimen for chemical testing. Correlation of urine pH with a patient’s condition aids in the diagnosis of disease (e.g., production of an alkaline urine despite a metabolic acidosis is characteristic of renal tubular acidosis). Individuals with a history of stone formation can monitor their urine pH and use this information to modify their diets if necessary. Highly alkaline urine of pH 8.0 to 9.0 can also interfere with chemical testing, particularly in protein determination.


Methods


Reagent strip tests

Commercial reagent strips, regardless of the manufacturer, are based on a double indicator system that produces varying color changes with pH (Equation 6.1). The indicator combinations produce color changes from yellow or orange (pH 5.0) to green (pH 7.0) to blue (pH 9.0). See Appendix B, Table B.1 for specific indicators used by different manufacturers.


IndOxidizeddyeyellow+H+ionsHIndReduceddyegreen to blue Equation 6.1


image Equation 6.1

The range provided on the strips is from pH 5.0 to pH 9.0 in 0.5- or 1.0-pH increments, depending on the manufacturer. No interferences with test results are known, and the results are not affected by protein concentration. However, erroneous results can occur from pH changes caused by (1) improper storage of the specimen with bacterial proliferation (a falsely increased pH); (2) contamination of the specimen container before collection (a falsely increased or decreased pH depending on the agent); or (3) improper reagent strip technique, causing the acid buffer from the protein test pad to contaminate the pH test area (a falsely decreased pH). See Table 6.4 for a summary of the pH principle, sensitivity, and specificity on selected reagent strip brands.



pH meter

Although the accuracy provided by a pH meter is not usually necessary, a pH meter is an alternative method for determining the urine pH. Various pH meters are available; the manufacturer’s operating instructions supplied with the instrument must be followed to ensure proper use of the pH meter and valid results. Nevertheless, the components involved in and the principle behind all pH meters are basically the same.


A pH meter consists of a silver–silver chloride indicator electrode with a pH-sensitive glass membrane connected by a salt bridge to a reference electrode (usually a calomel electrode, Hg–Hg2Cl2). When the indicator electrode is placed in urine, a difference in H+ activity develops across the glass membrane. This difference causes a change in the potential difference between the indicator and the reference electrodes. This voltage difference is registered by a voltmeter and is converted to a pH reading. Because pH measurement is temperature dependent and pH decreases with increasing temperature, it is necessary that the pH measurement be adjusted for the temperature of the urine during measurement. Most pH meters perform this temperature compensation automatically.


A pH meter is calibrated with the use of two or three commercially available standard buffer solutions. Accurate pH measurements require that the pH meter be calibrated using at least two different standards in the pH range of the test solution and that adjustment for the temperature of the test solution be made manually or automatically. In addition, the pH-sensitive glass electrode must be clean and maintained to prevent protein buildup or bacterial growth.


pH test papers

Various indicator papers with different pH ranges and sensitivities are commercially available. The indicator papers do not add impurities to the urine. In use, they produce sharp color changes for comparison with a supplied color chart of pH values.


Protein


Clinical Significance


Normal urine contains up to 150 mg (1 to 14 mg/dL) of protein each day. This protein originates from the ultrafiltration of plasma and from the urinary tract itself. Proteins of low molecular weight ([MW] <40,000) readily pass through the glomerular filtration barriers and are reabsorbed. Because of their low plasma concentration, only small quantities of these proteins appear in the urine. In contrast, albumin, a moderate-molecular-weight protein, has a high plasma concentration. This fact, combined with its ability (although limited) to pass through the filtration barriers, accounts for the small amount of albumin present in normal urine. Actually, less than 0.1% of plasma albumin enters the ultrafiltrate, and 95% to 99% of all filtered protein is reabsorbed. High-molecular-weight proteins (>90,000) are unable to penetrate a healthy glomerular filtration barrier. The end result is that the proteins in normal urine consist of about one-third albumin and two-thirds globulins. Among proteins that originate from the urinary tract itself, three are of particular interest: (1) uromodulin (also known as Tamm-Horsfall protein), which is a mucoprotein synthesized by the distal tubular cells and involved in cast formation; (2) urokinase, which is a fibrinolytic enzyme secreted by tubular cells; and (3) secretory immunoglobulin A,which is synthesized by renal tubular epithelial cells.2


The presence of an increased amount of protein in urine, termed proteinuria, is often the first indicator of renal disease. For most patients with proteinuria (prerenal and renal), the protein present at an increased concentration is albumin, although to varying degrees. Protein reabsorption by the renal tubules is a nonselective, competitive, and threshold-limited (Tm) process. Basically, when an increased amount of protein is presented to the tubules for reabsorption, the tubules randomly reabsorb the protein in a rate-limited process. As the quantities of proteins other than albumin increase and compete for tubular reabsorption, the amount of albumin excreted in the urine also increases. Proteinuria results from (1) an increase in the quantity of plasma proteins that are filtered, or (2) filtering of the normal quantity of proteins but with a reduction in the reabsorptive ability of the renal tubules. Early detection of proteinuria (i.e., albumin) aids in identification, treatment, and prevention of renal disease. However, protein excretion is not an exclusive feature of renal disorders, and other conditions can also present with proteinuria.


Proteinuria can be classified into four categories: prerenal or overflow proteinuria, glomerular proteinuria, tubular proteinuria, and postrenal proteinuria. This differentiation is based on a combination of protein origination and renal dysfunction; together, they determine the types and sizes of proteins observed in the urine (Table 6.5).



Table 6.5














Classification of Proteinuria
Proteinuria Classification Proteinuria Description Proteins Present Causes
Prerenal Overflow proteinuria: an increase in plasma low MW proteins leads to increased excretion in urine Normal proteins:

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Oct 18, 2022 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on Routine Urinalysis—the Chemical Examination

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