Abstract
The urinalysis is an essential component of the evaluation of patients with suspected parenchymal kidney disease and helps distinguish among the various causes of glomerular disease and acute or chronic kidney injury. It can be performed rapidly and at little expense. Chemical testing can be performed by individuals using the readily available dipstick technique or by high-throughput commercial laboratory instruments. However, automated examination of the spun urine sediment is not sufficiently reliable for accurate diagnosis. To properly identify blood cells, casts, crystals, lipid, infectious agents, and other formed elements, there is no substitute for manual examination of the resuspended spun urine sediment by an experienced observer.
Keywords
hematuria, proteinuria, red blood cells, dysmorphic red blood cells, white blood cells, tubular cells, pyuria, crystalluria, cylindruria, casts, hyaline casts, red blood cell casts, white blood cell casts, granular casts, waxy casts, broad casts, pigmented granular casts, tubular cell casts, degenerating cellular casts, crystals, acute tubular necrosis
The relatively simple chemical tests performed during routine urinalysis rapidly provide important information about a number of primary kidney and systemic disorders. The microscopic examination of the urine sediment is an indispensable part of the evaluation of patients with reduced glomerular filtration, proteinuria, hematuria, urinary tract infection, or nephrolithiasis, and the urine sediment provides valuable clues about the kidney parenchyma.
Urine dipstick tests can be readily automated, and most high-throughput clinical laboratories rely on computerized optical scanning or flow cytometry with automated instruments to perform microscopic urinalyses. Although these give reasonable results for detection of red blood cells (RBCs), white blood cells (WBCs), and squamous epithelial cells, they are unable to reliably identify critical elements such as renal tubular epithelial cells, oval fat bodies, crystals, or casts. Their accuracy for detection even of RBCs and WBCs falls with specimen aging, and sensitivity is reduced within as little as 2 hours after voiding. The interval between collection of a “routine” urine specimen, delivery to the lab, and processing may vary considerably.
When a primary kidney disorder is suspected, the automated urinalysis should be regarded only as a screening test. It does not supplant careful examination under the microscope of a specimen picked up promptly at the bedside, spun down, and examined at once. This task of careful review of the urine under the microscope must not be delegated; it should be performed personally by specialists experienced in examining the urine. Studies show both that a urinalysis performed by a nephrologist is more likely to aid in reaching a correct diagnosis than a urinalysis reported by a clinical chemistry laboratory and that urinalysis performed by physicians without special training is more often inaccurate. The features of a complete urinalysis are listed in Box 4.1 .
Appearance and Odor
Specific Gravity
Chemical Tests (Dipstick)
pH
Protein
Glucose
Ketones
Blood
Urobilinogen
Bilirubin
Nitrites
Leukocyte esterase
Microscopic Examination (Formed Elements)
Crystals: urate; calcium phosphate, oxalate, or carbonate; triple phosphate; cystine; drugs
Cells: leukocytes, erythrocytes, renal tubular cells, oval fat bodies, transitional epithelium, squamous cells
Casts: hyaline, granular, red blood cell, white blood cell, tubular cell, degenerating cellular, broad, waxy, lipid laden
Infecting organisms: bacteria, yeast, Trichomonas, nematodes
Miscellaneous: spermatozoa, mucous threads, fibers, starch, hair, and other contaminants
Specimen Collection and Handling
Urine should be collected with a minimum of contamination. A clean-catch midstream sample is preferred. If this is not feasible, bladder catheterization is appropriate in adults; the risk of inducing a urinary tract infection with a single in-and-out catheterization is negligible. Suprapubic aspiration is used in infants. In the uncooperative male patient, a clean, freshly applied condom catheter and urinary collection bag may be used. Urine in the collection bag of a patient with an indwelling bladder catheter is subject to stasis, but a sample suitable for examination may be collected by withdrawing urine from above a clamp placed on the tube that connects the catheter to the drainage bag.
The chemical composition of the urine changes with standing, and the formed elements within a urine sample degenerate over time. The urine is best examined when fresh, but a brief period of refrigeration is acceptable. Because bacteria multiply at room temperature, bacterial counts from unrefrigerated urine are unreliable. High urine osmolality and low pH favor cellular preservation, and these two characteristics of the first-voided morning urine give it particular value in cases of suspected glomerulonephritis. Some experts favor use of the second morning urine to avoid effects of overnight bladder stasis. However, the most important goal is examination without delay, regardless of what specimen is used.
Physical and Chemical Properties of the Urine
Appearance and Odor
Normal urine is clear with a faint yellow tinge due to the presence of urochrome. As the urine becomes more concentrated, its color deepens. Bilirubin, other pathologic metabolites, and a variety of drugs may discolor the urine or change its smell. Suspended erythrocytes, leukocytes, or crystals may render the urine turbid. Conditions associated with a change in the appearance or odor of the urine are listed in Table 4.1 .
| Color Change | Substances |
|---|---|
| White | Chyle, pus, calcium phosphate crystals, triple phosphate (struvite) crystals, propofol |
| Pink/red/brown | Erythrocytes, hemoglobin, myoglobin, porphyrins, beets, blackberries, senna, cascara, levodopa, methyldopa, deferoxamine, phenolphthalein and congeners, food colorings, metronidazole, phenacetin, anthraquinones, doxorubicin, phenothiazines, propofol, triple phosphate (struvite) crystals (salmon colored) |
| Yellow/orange/brown | Bilirubin, urobilin, phenazopyridine urinary analgesics, senna, cascara, mepacrine, iron compounds, nitrofurantoin, riboflavin, rhubarb, sulfasalazine, rifampin, fluorescein, phenytoin, metronidazole |
| Brown/black | Methemoglobin, homogentisic acid (alcaptonuria), melanin (melanoma), levodopa, methyldopa |
| Blue or green, green/brown | Biliverdin, Pseudomonas infection, dyes (methylene blue and indigo carmine), triamterene, vitamin B complex, methocarbamol, indican, phenol, chlorophyll, propofol, amitriptyline, triamterene |
| Purple staining of indwelling plastic urine collection devices | Infection with Escherichia coli, Pseudomonas, Enterococcus, others |
| Odor | Substance or Condition |
| Sweet or fruity | Ketones |
| Ammoniac | Urea-splitting bacterial infection |
| Fetid, pungent | Asparagus (sulfurous breakdown products) |
| Maple syrup | Maple syrup urine disease |
| Musty or mousy | Phenylketonuria |
| “Sweaty feet” | Isovaleric or glutaric acidemia, or excess butyric or hexanoic acid |
| Rancid | Hypermethioninemia, tyrosinemia |
Specific Gravity
The specific gravity of any fluid is the ratio of that fluid’s weight to the weight of an equal volume of distilled water. The urine specific gravity is a conveniently determined but inaccurate surrogate for osmolality. Specific gravities of 1.001 to 1.035 correspond to an osmolality range of 50 to 1000 mOsm/kg. A specific gravity near 1.010 connotes isosthenuria, with a urine osmolality matching that of plasma. Relative to osmolality, the specific gravity is elevated when dense solutes, such as protein, glucose, or radiographic contrast agents, are present.
Three methods are available for specific gravity measurement. The hydrometer is the reference standard but requires a sufficient volume of urine to allow flotation of the hydrometer and equilibration of the specimen to the calibrated temperature. The second method is based on the well-characterized relationship between urine specific gravity and refractive index. Refractometers calibrated in specific gravity units are commercially available and require only a drop of urine. Finally, the specific gravity may also be estimated by dipstick.
The specific gravity is used to determine whether the urine is concentrated. During a solute diuresis accompanying hyperglycemia, diuretic therapy, or relief of obstruction, the urine is isosthenuric. In contrast, with a water diuresis caused by overhydration or diabetes insipidus, the specific gravity is typically 1.004 or lower . In the absence of proteinuria, glycosuria, or iodinated contrast administration, a specific gravity of more than 1.018 implies preserved concentrating ability. Iodinated radiographic contrast is very dense, and if the specific gravity is supraphysiologic (i.e., >1.035), one should suspect that contrast is responsible. Measurement of specific gravity is useful in differentiating between prerenal azotemia and acute tubular necrosis (ATN) and in assessing the significance of proteinuria observed in a random voided urine sample. Because the protein indicator strip responds to the concentration of protein, the significance of a borderline reading depends on the overall urine concentration.
Routine Dipstick Methodology
The urine dipstick is a plastic strip to which absorbent tabs impregnated with chemical reagents have been affixed. The reagents in each tab are chromogenic. After timed development, the color is compared with a chart. Some reactions are highly specific. Others are affected by the presence of interfering substances or extremes of pH. Discoloration of the urine with bilirubin or blood may obscure the color changes.
pH
Test pads for pH use indicator dyes that change color with pH. The physiologic urine pH ranges from 4.5 to 8. The determination is most accurate if performed promptly, because growth of urea-splitting bacteria and loss of dissolved carbon dioxide raise the pH. In addition, bacterial metabolism of glucose may produce organic acids that lower pH. These strips are not sufficiently accurate to be used for the diagnosis of renal tubular acidosis. A specimen collected anaerobically under mineral oil should be promptly assayed with a pH electrode when precision is required.
Protein
Protein measurement uses the protein-error-of-indicators principle. The pH at which some indicators change color varies with the protein concentration of the bathing solution. Protein indicator strips are buffered at an acid pH near their color change point. Wetting them with a protein-containing specimen induces a color change. The protein reaction may be scored from trace to 4+ or by protein concentration. Their equivalence is approximately as follows: trace, 5 to 20 mg/dL; 1+, 30 mg/dL; 2+, 100 mg/dL; 3+, 300 mg/dL; 4+, greater than 2000 mg/dL. Highly alkaline urine, especially after contamination with quaternary ammonium skin cleansers or from patients who abuse sodium bicarbonate, may produce false-positive reactions by overwhelming the pH buffer of the chromogenic tab.
Protein strips are highly sensitive to albumin but less so to globulins, hemoglobin, or light chains. If light chain proteinuria is suspected, more sensitive assays should be used. With acid precipitation tests, an acid that denatures protein (i.e., sulfosalicylic acid) is added to the urine specimen, and the density of the precipitate is related to the protein concentration. Urine that is negative by dipstick but positive by sulfosalicylic acid precipitation is highly suspicious for the presence of light chains. Tolbutamide, high-dose penicillin, sulfonamides, and radiographic contrast agents may yield false-positive turbidimetric reactions. More sensitive and specific tests for light chains, such as immunoelectrophoresis or immunonephelometry, are preferred and necessary for confirmation and more definitive diagnosis.
If the urine is very concentrated, the presence of a modest protein reaction is less likely to correspond to significant proteinuria in a 24-hour collection or when assessed by spot urine protein-to-creatinine ratio. Even so, it is unlikely that a 3+ or 4+ reaction would be seen solely because of a high urine concentration or, conversely, that the urine would be dilute enough to yield a negative reaction despite significant proteinuria. The protein indicator used for routine dipstick analysis is neither sufficiently sensitive nor specific for albuminuria in the moderately increased (30 to 299 mg/g) or high normal range (10 to 29 mg/g).
Blood
Reagent strips for blood rely on the peroxidase activity of hemoglobin to catalyze an organic peroxide with subsequent oxidation of an indicator dye. Free hemoglobin produces a homogeneous color. Intact red cells cause punctate staining if present only in small quantity. False-positive reactions occur if the urine is contaminated with other oxidants, such as povidone-iodine, hypochlorite, or bacterial peroxidase. Ascorbate yields false-negative results. Myoglobin is also detected because it has intrinsic peroxidase activity. A urine sample that is positive for blood by dipstick analysis but shows no red cells on microscopic examination is suspect for myoglobinuria or hemoglobinuria. Pink discoloration of serum may occur with hemolysis, but free myoglobin is seldom present in a concentration sufficient to change the color of plasma. A specific assay for urine myoglobin confirms the diagnosis.
Specific Gravity
Specific gravity reagent strips measure ionic strength using indicator dyes with ionic strength-dependent dissociation constants (pKa). They do not detect glucose or nonionic radiographic contrast agents.
Glucose
Dipstick reagent strips are specific for glucose, relying on glucose oxidase to catalyze the formation of hydrogen peroxide, which then reacts with peroxidase and a chromogen to produce a color change. High concentrations of ascorbate or ketoacids reduce test sensitivity; however, the degree of glycosuria occurring in diabetic ketoacidosis is sufficient to prevent false-negative results despite ketonuria.
Ketones
Ketone reagent strips depend on the development of a purple color after acetoacetate reacts with nitroprusside. Some strips can also detect acetone, but none react with β-hydroxybutyrate. False-positive results may occur in patients who are taking levodopa or drugs such as captopril or mesna that contain free sulfhydryl groups.
Urobilinogen
Urobilinogen is a colorless pigment produced in the gut from the metabolism of bilirubin. Some is excreted in feces, and the rest is reabsorbed and excreted in the urine. In obstructive jaundice, bilirubin does not reach the bowel, and urinary excretion of urobilinogen is diminished. In other forms of jaundice, urobilinogen is increased. The urobilinogen test is based on the Ehrlich reaction in which diethylaminobenzaldehyde reacts with urobilinogen in acid medium to produce a pink color. Sulfonamides may produce false-positive results, and degradation of urobilinogen to urobilin may yield false-negative results. Better tests are available to diagnose obstructive jaundice.
Bilirubin
Bilirubin reagent strips rely on the chromogenic reaction of bilirubin with diazonium salts. Conjugated bilirubin is not normally present in the urine. False-positive results may be observed in patients receiving chlorpromazine or phenazopyridine. False-negative results occur in the presence of ascorbate.
Nitrite
The nitrite screening test for bacteriuria relies on the ability of gram-negative bacteria to convert urinary nitrate to nitrite, which activates a chromogen. False-negative results occur with infection with enterococcus or other organisms that do not produce nitrite, when ascorbate is present, or when urine has not been retained in the bladder long enough (approximately 4 hours) to permit sufficient production of nitrite from nitrate.
Leukocyte Esterase
Granulocyte esterases can cleave pyrrole amino acid esters, producing free pyrrole that subsequently reacts with a chromogen. The test threshold is 5 to 15 WBCs per high-power field (WBCs/HPF). False-negative results occur with glycosuria, high specific gravity, cephalexin or tetracycline therapy, or excessive oxalate excretion. Contamination with vaginal material may yield a positive test result without true urinary tract infection.
Microalbumin Dipsticks
Albumin-selective dipsticks are available for screening for “microalbuminuria” (moderately increased albuminuria in the range of 30 to 299 mg/g). The most accurate screening occurs when first morning specimens are examined as exercise can increase albumin excretion. One type of dipstick uses colorimetric detection of albumin bound to gold-conjugated antibody. Normally, the urine albumin concentration is less than the 20 µg/L detection threshold for these strips. Unless the urine is very dilute, a patient with no detectable albumin by this method is unlikely to have microalbuminuria. However, because urine concentration varies widely, this assay has the same limitations as any test that only measures concentration. It is useful only as a screening test, and more formal testing is required if albuminuria is detected.
A second type of dipstick has tabs for measurement of both albumin and creatinine concentration, permitting estimation of the albumin-to-creatinine ratio. In contrast to the other dipstick tests described in this chapter, these strips cannot be read by simple visual comparison with a color chart. An instrument is required, but this system is suitable for point-of-care testing. When present on more than one determination, an albumin-to-creatinine ratio of 30 to 300 µg/mg signifies moderately increased albuminuria. Details on the interpretation of urine albumin concentration are provided in Chapters 5 and 26 .
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