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Chemical Analysis of Urine 37 LWBK461-c04_p35-54.qxd 11/17/09 7:57 PM Page 35 Aptara Inc CHAPTER ChemicalChemical AnalysisAnalysis ofof UrineUrine 4 Key Terms ACETEST ASCORBATE (ASCORBIC ACID) BILIRUBIN CHROMOGENS CLINITEST GLYCOSURIA HEMATURIA HEMOGLOBINURIA ICTOTEST KETONES Learning Objectives LEUKOCYTE ESTERASE For Each Chemical Test Performed by Dipstick Methodology MYOGLOBIN NITRITE 1. Describe the principle and procedure. 2. Compare and contrast reagent strip characteristics among manufacturers. pH 3. Interpret results. PROTEIN 4. Define expected normal values. PROTEIN ERROR OF INDICATORS 5. Suggest causes for abnormal findings. PROTEINURIA 6. Identify sources of error. REAGENT STRIP 7. Suggest appropriate confirmatory tests. REDUCING SUBSTANCE 8. Correlate results of chemical tests with those of physical examination. RUN-OVER 9. Predict findings of microscopic examination. TAMM–HORSFALL PROTEIN UROBILINOGEN For the Confirmatory Urine Tests (Acetest, Clinitest, Ictotest) 10. Describe the principle and procedure. 11. Interpret results. 12. Recognize sources of error. 13. Suggest appropriate clinical applications. 35 LWBK461-c04_p35-54.qxd 11/17/09 7:57 PM Page 36 Aptara Inc 36 Graff’s Textbook of Routine Urinalysis and Body Fluids he routine urinalysis includes chemical testing for pH, room temperature before testing. The procedure for using protein, glucose, ketones, occult blood, bilirubin, uro- the dipstick is as follows: bilinogen, nitrite, leukocyte esterase, and strip test T 1. Completely dip the test areas of the strip in fresh, well- method for specific gravity. The urinalysis offered by labo- mixed, uncentrifuged urine and remove immediately. ratories depends on the type of dipstick that is used. In Care should be taken not to touch the test areas. addition, most laboratories routinely screen for reducing 2. Remove the excess urine from the stick by touching substances as part of the routine urinalysis for children the edge of the strip to the urine container. Follow the 2 years old and younger. These procedures are either quali- manufacturer’s requirement for maintaining the tative (positive or negative) or semiquantitative (e.g., trace reagent strip in either a horizontal or vertical through 4ϩ) measurements. Since the introduction of single- position. and multiple-test reagent strips, test tapes, and tablets, the 3. At the correct times, compare the test areas with the chemical screening of the urine has become a sensitive and corresponding color charts on the container. The rapid procedure. Completion of urine chemistry using strip should be read in good lighting for accurate reagent test strips occurs in 2 minutes. Several brands of color comparison. dipsticks are available worldwide. A comparison of some of 4. Record results as prescribed by your laboratory’s the strips available from various manufacturers is made in protocol. this chapter. A reagent strip, also called a dipstick, is a narrow strip of Several brands of urine chemistry dipsticks are com- plastic with small pads attached to it. Each pad contains pared in this text. The reagents used for these dipsticks vary reagents for a different reaction, thus allowing for the according to manufacturer. The reagents for each parame- simultaneous determination of several tests. The colors ter measured by these manufacturers along with their sen- generated on each reagent pad vary according to the concen- sitivities are listed in tables that appear with the discussion tration of the analyte present. Colors generated by each pad of each parameter. Although examples of each parameter’s are visually compared against a range of colors on brand- color reactions are also included, they portray the results specific color charts. Color charts for one brand of reagent obtained by only one manufacturer. Chemical reaction col- strips discussed in this text is included in Appendix C. ors vary slightly as do the timing of the reactions. Color Figure 4-1 illustrates a typical urine chemistry reagent strip charts for one brand of reagent strips discussed in this text is (dipstick).1 included in Appendix C. Always review and follow the man- The manual method for using a reagent strip to test ufacturers’ latest directions, as improvements to the reagent urine calls for dipping the entire strip into the specimen strips may have been made for more recently manufactured and withdrawing it in one continuous motion while remov- lot numbers of strips. ing excess urine by dragging across the edge of the specimen Even with the widespread use of the rapid and conven- container.2 A critical requirement is that the reactions be ient screening procedures, it is still necessary to understand read at the prescribed time after dipping and then com- the basic principles of the tests as well as the correct tech- pared closely with the color chart provided by the manufac- nique to be used. This chapter includes a clinical explana- turer. To obtain accurate and reliable results with the dip- tion of the chemical constituents most often tested in sticks, certain precautions must be taken to help maintain urine, the principles behind the tests, some causes for the reactivity of the reagents. The strips must not be abnormal results, and use of confirmatory procedures. exposed to moisture, direct sunlight, heat, or volatile sub- stances; and they should be stored in their original contain- ers. The container should not be kept in the refrigerator nor URINARY pH exposed to temperatures over 30ЊC. Each vial or bottle contains a desiccant, but the strips should still not be exposed to moisture. Remove only the number of strips One of the functions of the kidney is to help maintain needed at the time of testing and then tightly close the con- acid–base balance in the body. To maintain a constant pH tainer. If the color blocks on the strip do not resemble the (hydrogen ion concentration) in the blood (about 7.40), the negative blocks on the color chart or if the expiration date kidney must vary the pH of the urine to compensate for diet on the container has past, discard the strips. and products of metabolism. This regulation occurs in the Urine should be tested at room temperature. If the urine distal portion of the nephron with the secretion of both specimen has been refrigerated, it should be brought to hydrogen and ammonia ions into the filtrate, and the reab- ϩ sorption of bicarbonate. If sufficient hydrogen ions (H ) are secreted into the tubule, all of the bicarbonate present ϩ Urobilin- Specific will be reabsorbed, but if fewer H are secreted or if an Leukocytes Nitrite Protein pH Blood Ketone Bilirubin Glucose Hold this end ogen gravity excess of bicarbonate is present, some of the bicarbonate 3 ϩ Figure 4-1. Illustration of Multistix 10 SG. (Modified from Tarrytown, will be excreted in the urine. The continued secretion of H NY: Bayer Corporation Diagnostics Division, 1996.1) after all bicarbonate has been reabsorbed will drop the pH LWBK461-c04_p35-54.qxd 11/17/09 7:57 PM Page 37 Aptara Inc Chapter 4—Chemical Analysis of Urine 37 of the filtrate and result in an acidic urine. The secretion of the test pads and underlying absorbent papers in place on the ϩ H in the tubule is regulated by the amount present in the plastic strip.5 The mesh allows for even diffusion of the urine body. If there is an excess of acid in the body (acidosis), on the test pads, and the underlying paper absorbs excess ϩ more H will be excreted and the urine will be acid. When urine to prevent “run-over.” If pH is the only test needed to be ϩ there is an excess of base in the body (alkalosis), less H will done on a urine specimen, litmus paper or Nitrazine paper be excreted and the urine will be alkaline. The hydrogen can also be used to obtain an approximate reading. ϩ ions in the urine are excreted as either free H , in associa- tion with a buffer such as phosphate, or bound to ammonia as ammonium ions. The pH of the urine is determined by ϩ the concentration of the free H . PROTEIN Because pH is the reciprocal of the hydrogen ion concen- ϩ tration, as the H concentration increases, the pH decreases ϩ The presence of increased amounts of protein in the urine or becomes more acidic. As the H concentration decreases, can be an important indicator of renal disease. It may be the the pH increases or becomes more alkaline. The pH of the first sign of a serious problem and may appear long before urine may range from 4.6 to 8.0 but averages around 6.0, so other clinical symptoms. There are, however, physiologic it is usually slightly acidic. There is no abnormal range as conditions such as exercise and fever that can lead to such, since the urine can normally vary from acid to alka- increased protein excretion in the urine in the absence of line. For this reason, it is important for the physician to cor- renal disease. There are also some renal disorders in which relate the urine pH with other information to determine proteinuria is absent. whether there is a problem. Metabolic and renal disorders In the normal kidney, only a small amount of low– that affect urine pH are discussed in Chapter 5. molecular weight protein is filtered at the glomerulus. The structure of the glomerular membrane prevents the passage REAGENT TEST STRIPS of high–molecular weight proteins including albumin (mol wt ϭ 69,000). After filtration, most of the protein is reab- All brands of dipsticks discussed in this chapter use the sorbed in the tubules with less than 150 mg/24 h (or 20 same two indicators, methyl red and bromthymol blue, and mg/dL) being excreted. In a child, the normal excretion is 2 6 measure a range of pH from 5.0 to 8.5.
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