Giovanni B. Fogazzi, Giuseppe Garigali Urinalysis is one of the key tests to evaluate kidney and urinary tract disease. When a patient is first seen by a nephrologist, urinalysis should always be performed. Dipsticks are the most widely used method for urinalysis, but the nephrologist should be aware of their limitations. Urine sediment examination is an integral part of urinalysis, which is performed routinely in general clinical laboratories. Ideally, however, urine microscopy should be performed by trained nephrologists rather than clinical laboratory personnel, who are at times unable to identify important elements,1 and who are not always aware of the clinical correlates of the findings.2 This chapter describes the main aspects of urinalysis, including urine collection, evaluation of physical and chemical features of urine, and urine microscopy. The way urine is collected and handled can greatly influence the results (Box 4-1). Written instructions for performing a urine collection should be given to the patient. First, strenuous physical exercise (e.g., running, soccer) should be avoided for at least 24 hours before the collection to avoid exercise-induced proteinuria and hematuria or cylindruria. In women, urinalysis should also be avoided during menstruation because blood contamination can easily occur. If a midstream sample of the first morning urine is used, lysis of cells and casts may occur in the bladder overnight, which may lead to false-negative results at urine sediment examination. For this reason, for renal patients, we suggest performing a combined dipstick and urine microscopy on the second morning urine. For the measurement of 24-hour protein excretion, a 24-hour urine collection is needed. Errors caused by improper timing and missed samples can lead to overcollection or undercollection of urine. Errors can be minimized by giving the patient simple written instructions. Written instructions should also be provided for other types of urine collection, such as that needed for the evaluation of orthostatic proteinuria, which implies the collection of one sample produced while the patient has been recumbent for some hours, and another sample produced while the patient has been standing. Spot urine samples are widely recommended3 because they are easy to obtain, although timed urines are still recommended by some authorities (see Chapter 80). After the washing of hands, women should spread the labia of the vagina and men withdraw the foreskin of the glans. The external genitalia are washed and wiped dry with a paper towel, and the “midstream” urine is collected after the first portion is discarded. The same procedures can also be used for children. For small infants, bags for urine are often used, even though these carry a high probability of contamination. A suprapubic bladder puncture may occasionally be necessary. In special situations, urine can also be collected through a bladder catheter, although the catheter may cause hematuria. Permanent indwelling catheters are often associated with bacteriuria, leukocyturia, hematuria, and candiduria. The container for urine should be provided by the laboratory or bought in a pharmacy. It should be clean, have a capacity of at least 50 to 100 ml, and have a diameter opening of at least 5 cm to allow easy collection. It should have a wide base to avoid accidental spillage and should be capped. The label should identify the patient as well as the hour of urine collection. Several elements (but especially leukocytes) can lyse rapidly after collection, thus ideally the sample should be handled and examined as soon as possible. In everyday practice, we suggest the samples be analyzed within 3 hours from collection. If this is not possible, refrigeration of specimens at +4° to +8° C assists preservation but may cause precipitation of phosphates or urates, which can hamper examination. Alternatively, chemical preservatives such as formaldehyde or glutaraldehyde can be used. The color of normal urine ranges from pale to dark yellow and amber, depending on the concentration of the urochrome. Abnormal changes in color can be caused by pathologic conditions, drugs, or foods. The most frequent pathologic conditions that can cause color changes of the urine are gross hematuria, hemoglobinuria, or myoglobinuria (pink, red, brown, or black urine); bilirubinuria (dark-yellow to brown urine); and massive uric acid crystalluria (pink urine). Less frequent causes are urinary infection, mainly from Klebsiella spp., Proteus mirabilis, Escherichia coli, Providencia stuartii, or Enterococcus spp. in patients with permanent bladder catheter (purple urine, sometimes called “purple urine bag syndrome”)4; chyluria (white milky urine); and porphyrinuria (associated with the excretion in the urine of porphobilinogen) and alkaptonuria (red urine turning black on standing). The main drugs responsible for abnormal urine color are rifampin (yellow-orange to red urine); desferrioxamine (pinkish urine); phenytoin (red urine); chloroquine and nitrofurantoin (brown urine); triamterene, propofol, and blue dyes of enteral feeds (green urine); methylene blue (blue urine); and metronidazole, methyldopa, and imipenem-cilastatin (darkening on standing). Among foods are beetroot (red urine), senna and rhubarb (yellow to brown or red urine), and carotene (brown urine). Normal urine is transparent. Urine can be turbid because of a high concentration of any urine particle, especially cells, crystals, and bacteria. The most frequent causes of turbidity are urinary tract infection, heavy hematuria, and contamination of urine from genital secretions. The absence of turbidity is not a reliable criterion by which to judge a urine sample because pathologic urine can be transparent. A change in urine odor may be caused by the ingestion of some foods, such as asparagus. A pungent odor, caused by the production of ammonia, is typical of most bacterial urinary tract infection, whereas there is often a sweet or fruity odor with ketones in the urine. Some rare conditions confer a characteristic odor to the urine. These include maple syrup urine disease (maple syrup odor), phenylketonuria (musty or mousy odor), isovaleric acidemia (sweaty feet odor), and hypermethioninemia (rancid butter or fishy odor). The relative density parameter can be measured by specific gravity or osmolality. Specific gravity (SG) refers to the weight of a volume of urine compared with the weight of the same volume of distilled water and depends on the mass and number of the dissolved particles. SG is most frequently evaluated by dipstick, which measures the ionic concentration of urine. In the presence of ions, protons are released by a complexing agent and produce a color change in the indicator bromthymol blue from blue to blue-green to yellow. Underestimation occurs with urine pH above 6.5, whereas overestimation is found with urine protein concentration above 7.0 g/L. Because nonionized molecules, such as glucose and urea, are not detected by dipstick, this method does not strictly correlate with the results obtained by refractometry and osmolality. Refractometry measures SG through the refraction of light while it passes through a drop of urine on a glass plate. This measures the number of solutes per unit volume and measures all solutes rather than just ionic substances. Therefore, refractometry is more accurate than dipstick, despite being influenced by urine temperature, although temperature-compensated refractometers are available. Refractometers are inexpensive, simple to use, and have the major advantage of requiring only 1 drop of urine. For these reasons, we suggest the use of refractometry for everyday practice. An SG of 1.000 to 1.003 is consistent with marked urinary dilution, as observed in patients with diabetes insipidus or water intoxication. SG of 1.010 is often called isosthenuric urine because it is of similar SG (and osmolality) to plasma, so it is often observed in conditions in which urinary concentration is impaired, such as acute tubular necrosis (ATN) and chronic kidney disease. SG above 1.040 almost always indicates the presence of some extrinsic osmotic agent, such as radiocontrast. Osmolality is measured by an osmometer, which evaluates the freezing-point depression of a solution and supplies results as milliosmoles per kilogram (mOsm/kg) of water. Osmolality depends only on the number of particles present and is not influenced by urine temperature or protein concentrations. However, high glucose concentrations significantly increase osmolality (10 g/l of glucose = 55.5 mOsm/l). The measurement of osmolality is more reliable than SG by either dipstick or refractometry for the evaluation of pathologic urine. Chemical characteristics of urine are most frequently evaluated by dipstick. Dipsticks have the advantages of simplicity, low cost, and stability. Disadvantages include qualitative or semiquantitative results only, susceptibility to interference by substances, and urine discoloration. When the reading is visual and not by automated instruments, the interval between removal of the dipstick from urine and the reading of results indicated by the manufacturer must be respected to avoid false results. Sensitivity and specificity of dipsticks greatly differ among studies and depend on the brand used and different clinical conditions and patient populations investigated. False results can also be caused by the use of time-expired dipsticks. Table 4-1 summarizes the main false-negative and false-positive results that can occur with urine dipstick testing. Table 4-1 Urine dipstick testing. Main false-negative and false-positive results of urine dipsticks. False results may also occur when time-expired dipsticks are used. The pH is determined by dipsticks that cover the pH range of 5.0 to 8.5 or to 9.0. With use of dipsticks, significant deviations from true pH are observed for values below 5.5 and above 7.5. Therefore, a pH meter with a glass electrode is mandatory if an accurate measurement is necessary. Urine pH reflects the presence of hydrogen ions (H+), but this does not necessarily reflect the overall acid load in the urine because most of the acid is excreted as ammonia. A low pH is often observed with metabolic acidosis (in which acid is secreted), with high-protein meals (which generate more acid and ammonia), and with volume depletion (in which aldosterone is stimulated, resulting in an acid urine). Indeed, low urine pH may help distinguish pre-renal acute kidney injury (AKI) from ATN, which is typically associated with a higher pH. High pH is often observed with renal tubular acidosis (especially distal, type 1; see Chapter 12), with vegetarian diets (caused by minimal nitrogen and acid generation), and with infection with urease-positive organisms (e.g., Proteus) that generate ammonia from urea. Measurement of urine pH is also needed for the interpretation of urinalysis (see Leukocyte Esterase and Urine Microscopy). Hemoglobin is detected by a dipstick on the basis of the pseudoperoxidase activity of the heme moiety of hemoglobin, which catalyzes the reaction of a peroxide and a chromogen to form a colored product. The presence of hemoglobin is shown as green spots, which result from intact erythrocytes, or as a homogeneous, diffuse green pattern. This can result from marked hematuria because of the high number of erythrocytes that cover the whole pad surface; from lysis of erythrocytes favored by delayed examination, alkaline urine pH, or low SG; or from hemoglobinuria secondary to intravascular hemolysis. False-negative results are mainly caused by (1) ascorbic acid, a strong reducing agent, which can result in low-grade microscopic hematuria being completely missed,5 and (2) high SG. The most important causes of false-positive results are myoglobinuria, resulting from rhabdomyolysis, and a high concentration of bacteria with pseudoperoxidase activity (Enterobacteriaceae, staphylococci, and streptococci).6 Glucose is also often detected by dipstick. With glucose oxidase as catalyst, glucose is first oxidized to gluconic acid and hydrogen peroxide. Through the catalyzing activity of a peroxidase, hydrogen peroxide then reacts with a reduced colorless chromogen to form a colored product. This test detects concentrations of 0.5 to 20 g/l. When more precise quantification of urine glucose is needed, enzymatic methods such as hexokinase must be used. False-negative results with glucose detection occur in the presence of ascorbic acid and bacteria. False-positive findings may be observed in the presence of oxidizing detergents. Although there is no consistent definition of proteinuria,7 it is accepted that physiologic proteinuria does not exceed 150 mg/24 h for adults and 140 mg/m2 for children. Three different approaches can be used for the evaluation of proteinuria, as described next. The albumin dipstick test is based on the presence of protein in a buffer causing a change in pH proportional to the concentration of protein itself. The dipstick changes its color, from pale green to green and blue, according to the pH changes induced by the protein. The dipstick for protein is sensitive to albumin but has a very low sensitivity to other proteins, such as tubular proteins and light-chain immunoglobulins; thus the dipstick will not detect the overflow proteinuria that can occur in myeloma. Moreover, the detection limit is 0.25 to 0.3 g/l, which may be too high to identify the early phases of kidney disease (i.e., microalbuminuria) and is influenced by hydration status (false-negative results may occur at low urine SG, and vice versa) and urine pH (false-positive results at strongly alkaline pH). Also, dipstick supplies only a semiquantitative measurement of urine albumin, which is expressed on a scale from 0 to +++ or ++++.7 Some manufacturers also supply numerical results, although these represent only approximate quantitative measurements. Thus, for accurate quantification, other methods are needed. Recently, a creatinine test pad has been added to some dipsticks, which supplies a protein-creatinine ratio (PCR) and reduces the variability caused by changing diuresis and urine dilution.8 The 24-hour urine collection for protein excretion remains the reference (gold standard) method. It is based on chemical assay (e.g., biuret or Folin-Lowry reaction), turbidimetric technique (e.g., trichloroacetic acid, benzethonium chloride, ammonium chloride), or dye-binding technique (e.g., ponceau S, Coomassie brilliant blue G-250, pyrogallol red molybdate), which quantify total proteins rather than simply albumin. The 24-hour protein excretion averages the variation of proteinuria caused by the circadian rhythm and is the most accurate for monitoring of proteinuria during treatment. However, it can be impractical in some settings (e.g., children, outpatients, elderly patients) and is subject to error from overcollection or undercollection. For this reason, we give our patients written, simple but definitive instructions on how to collect urine (see earlier discussion). This PCR is obtained by the ratio between urine protein excretion (measured by methods in 24-Hour Protein Excretion) and creatinine excretion, expressed as mg/mg or mg/mmol. PCR represents a practical alternative to the 24-hour urine collection because it is easy to obtain and is not influenced by variation in water intake or rate of diuresis.3 Also, the same sample can also be used for microscopic investigation. A close correlation between the PCR in a random urine sample and the 24-hour protein excretion has been demonstrated in a wide range of patients,7,9 including those with different types of glomerulonephritis (GN) evaluated longitudinally during treatment.10 However, the results may be influenced by a reduced creatinine excretion because of reduced muscle mass. Thus, in elderly and female patients, PCR values can be higher than in young men. Another factor to be considered is the timing of the sample, which is influenced by the daily circadian fluctuation of protein excretion in the presence of minimal corresponding variation of creatinine excretion. Thus, the best estimates are probably obtained with morning samples, but not the first void.11 Some consider that a normal PCR is sufficient to rule out pathologic proteinuria, but that an elevated PCR should be confirmed and quantified with a 24-hour collection.12 Other investigators have found poor correlation between PCR and 24-hour proteinuria at high levels of protein excretion,10 or that PCR is an unreliable method to monitor some patients with lupus nephritis13,14 (see also Chapter 80). A possible alternative to PCR is the measurement of albumin-creatinine ratio (ACR), especially to screen and monitor diabetic patients.9 However, with ACR false-negative results may occur,15 as a consequence of the variable proportion of albumin present in the urine, which may depend on the underlying renal disease. An elevated PCR with a negative ACR for example suggests the diagnosis of myeloma. The ratio of urinary albumin to total protein excretion (urine albumin/protein ratio, uAPR) has recently been proposed as a method to distinguish proteinuric patients with a pure glomerular disease from patients with a glomerular disease associated with tubulointerstitial damage or with a tubulointerstitial nephropathy. However, uAPR is not yet validated for routine clinical practice.16,17 Defined as the presence of albumin in the urine in a range of 30 to 299 mg/24 h, microalbuminuria identifies diabetic patients at increased risk of developing overt diabetic nephropathy. Also, in the general population, microalbuminuria identifies patients at increased risk of chronic kidney disease, cardiovascular morbidity, and overall mortality. The 24-hour urine collection, initially considered the gold standard method for the detection of microalbuminuria, currently has been replaced by the use of early-morning urine, which minimizes the changes caused by diurnal volume variations. A number of semiquantitative dipstick tests are available to screen for microalbuminuria. Once microalbuminuria is found by dipstick, a standard quantitative method is then used for confirmation, usually immunoassay.18 Because of its great simplicity, immunoturbidometry is the method most frequently used. Low-molecular-weight tubular proteins such as α1-microglobulin, retinol-binding protein, and β2-microglobulin are identified by a qualitative analysis of urine proteins, using electrophoresis on cellulose acetate or agarose after protein concentration or using very sensitive stains such as silver and gold. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) can be used to identify tubular proteins in the urine of patients with glomerular diseases, which may have therapeutic and prognostic implications.19 The Bence Jones protein indicates the presence in the urine of free immunoglobulin light chains, as occurs in patients with monoclonal gammopathies. Bence Jones proteinuria is revealed by urine electrophoresis, whereas light-chain identification requires urine immunofixation.20 Selectivity can be assessed in nephrotic patients by the ratio of the clearance of IgG (molecular weight of 160,000 d) to the clearance of transferrin (88,000 d).21 Although now used infrequently, highly selective proteinuria (ratio <0.1) in nephrotic children suggests the diagnosis of minimal change disease and predicts corticosteroid responsiveness. The leukocyte esterase dipstick test evaluates the presence of leukocytes based on the activity of an indoxyl esterase released from lysed neutrophil granulocytes. Leukocyte esterase may be positive when microscopy is negative and when leukocytes are lysed, because of low relative density, alkaline pH, or a delay in sample handling and examination. False-negative results derive from high glucose (≥20 g/l) or high protein (≥5 g/l) concentration or from the presence of antibiotics such as cephalothin and tetracycline (strong inhibition), cephalexin (moderate inhibition), or tobramycin (mild inhibition). The sensitivity is also reduced by high SG because this prevents leukocyte lysis. False-positive results may occur when formaldehyde is used as a urine preservative and, according to one report, from the presence in the urine of imipenem, meropenem, or clavulanate.22 The dipstick nitrites test detects bacteria that reduce nitrates to nitrites by nitrate reductase activity. This includes most gram-negative uropathogenic bacteria, but not Pseudomonas, Staphylococcus albus, or Enterococcus. A positive test result also occurs with a diet rich in nitrates (vegetables), which form the substrate for nitrite production, and sufficient bladder incubation time. Thus, not surprisingly, the sensitivity of the dipstick nitrites test is low, but specificity is high.23 Measurement of urinary urobilinogen and bilirubin concentrations has lost its clinical value in the detection of liver disease after the introduction of serum tests of liver enzyme function. The ketone dipstick tests for acetoacetate and acetone (but not β-hydroxybutyrate), which are excreted into urine during diabetic acidosis or during fasting, vomiting, or strenuous exercise. It is based on the reaction of the ketones with nitroprusside. The second urine specimen of the morning should be collected because it avoids the lysis of particles that can occur in the bladder overnight (see Urine Collection and Box 4-1). We centrifuge an aliquot of urine within 3 hours from collection and concentrate it by removal of a fixed aliquot of supernatant urine. After this, the sediment is resuspended with a Pasteur pipette, and a fixed aliquot is transferred to the slide and prepared using a coverslip with a fixed surface.
Urinalysis
Definition
Urine Collection
Physical Characteristics
Color
Turbidity
Odor
Relative Density
Chemical Characteristics
Urine Dipstick Testing
Constituent
False-negative Results
False-positive Results
Specific gravity (SG)
Urine pH >6.5
Urine protein >7.0 g/l
pH
Reduced values in presence of formaldehyde
—
Hemoglobin
Ascorbic acid
High SG of urine
Formaldehyde (0.5 g/l) used to preserve samples
Myoglobin
Microbial peroxidases
Glucose
Ascorbic acid
Bacteria
Oxidizing detergents
Albumin
Immunoglobulin light chains
Tubular proteins
Globulins
Urine pH ≥9.0
Quaternary ammonium detergents
Chlorhexidine
Polyvinylpyrrolidone
Leukocyte esterase
Glucose ≥20.0 g/l
Protein >5.0 g/l
Cephalothin (+++)
Tetracycline (+++)
Cephalexin (++)
Tobramycin (+)
High SG of urine
Formaldehyde (0.4 g/l)
?Imipenem
?Meropenem
?Clavulanate
Abnormally colored urine
Nitrites
Bacteria that do not reduce nitrates to nitrites
No vegetables in diet
Short bladder incubation time
Abnormally colored urine
Ketones
Improper storage
Free sulfhydryl groups (e.g., captopril)
Levodopa
Abnormally colored urine
pH
Hemoglobin
Glucose
Protein
Albumin Dipstick
24-Hour Protein Excretion
Protein-Creatinine Ratio on Random Urine Sample
Specific Proteins
Microalbuminuria
Tubular Proteins
Bence Jones Proteinuria
Selectivity of Proteinuria
Leukocyte Esterase
Nitrites
Bile Pigments
Ketones
Urine Microscopy
Methods
Related posts:
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
Urinalysis
Chapter 4
