The appearance of the specimen is usually reported only if it is abnormal.
Red: Blood; porphyria; occasionally urates, phenolphthalein, or dihydroxyanthraquinone (Dorbane) (laxative use)
Brown: Blood (acid hematin); alkaptonuria (urine turns brownish on standing); melanin (may be brown and turn black on standing)
Dark orange: Bile, pyridium (a urinary tract disinfectant)
pH
Normal urine pH is 5.0-6.0 (4.5-8.0); it is over 6.0 in alkalosis or, rarely, if the acidifying mechanism of the kidney fails (renal acidosis syndrome) or is poisoned (carbonic anhydrase inhibitors). Proteus infections typically will alkalinize. Finally, urine standing at room temperature will often slowly become alkaline due to bacterial growth. A pH determination is of minor importance except in a few circumstances.
Specific gravity
Specific gravity will be discussed in greater length in Chapter 13 (renal function). It is important for several reasons: (1) it is one parameter of renal tubular function; (2) inability to concentrate may accompany certain diseases with otherwise relatively normal tubular function, such as diabetes insipidus or, occasionally, severe hyperthyroidism and sickle cell anemia; and (3) it affects other urine tests; a concentrated specimen may give higher results for substances such as protein than a dilute specimen, although the amount of substance excreted per 24 hours may be the same in both cases. That is, the same amount of substance assayed in a small quantity of fluid (high specific gravity) may appear more than if present in a large dilute urine volume (low specific gravity).
Specific gravity is an estimate of relative density (for fluids, the proportion of dissolved solids in a defined volume of solvent) and can be measured in various ways. The oldest was by hydrometer (weighted flotation device), whose reproducibility was poor and which was relatively inaccurate. Most laboratories use some version of a refractometer (e.g., a total solids meter), which is fairly reproducible and accurate, but which is affected by moderate amounts of protein, glucose, or x-ray contrast media. More recently, dipstick specific gravity tests have become available based on changes in urine ion content that produce a pH change of the polyelectrolyte content of the reagent pad, which, in turn, induces color changes of an indicator (bromthymol blue). This method is read in increments of 0.005 units. Several evaluations suggest that the dipstick results will agree within ±0.005 units of total solids meter values about 85% (range 72%-90%) of the time. This suggests that the dipstick method is somewhat less accurate than the total solids meter. Whether the dipstick method is useful depends on whether a difference of 0.004-0.005 from the total solids meter value is considered clinically acceptable. The dipstick is affected by moderate changes in pH or protein but not by glucose or x-ray contrast media.
Protein
In health the glomerular membrane prevents most of the relatively large protein molecules of the blood from escaping into the urine. A small amount does filter through, of which a small fraction is reabsorbed by the tubules, and the remainder (up to 0.1 gm/24 hours) is excreted. These amounts are normally not detected by routine clinical methods. One of the first responses of the kidney to a great variety of clinical situations is an alteration in the glomerular filtrate. To a lesser extent, a mere increase in glomerular filtration rate (GFR) may increase normal protein excretion. Since albumin has a relatively small molecular size, it tends to become the dominant constituent in proteinuria but rarely so completely as to justify the term “albuminuria” in a pure sense. Depending on the clinical situation, the constituents of proteinuria may or may not approach the constituents of plasma. Besides an excess of normal plasma proteins, at times abnormal proteins may appear in the urine. The most important of these is Bence Jones protein, which is excreted in up to 50% of patients with multiple myeloma.
Etiologies of proteinuria. To interpret proteinuria, one must know not only the major conditions responsible but also the reason for production of excess urine protein. A system such as the following one is very helpful in this respect; proteinuria is classified according to the relationship of its etiology to the kidney and also the mechanism involved.
A. Functional: not associated with easily demonstrable systemic or renal damage.
1. Severe muscular exertion. This cause is becoming more important with the current popularity of sports and jogging.
2. Pregnancy.
3. Orthostatic proteinuria. (This term designates slight to mild proteinuria associated only with the upright position. The exact etiology is poorly understood, with usual explanations implicating local factors that cause renal passive congestion when the patient is in an upright position. Orthostatic proteinuria is easily diagnosed by comparing a specimen of early morning urine, produced entirely while the patient is asleep, with one obtained later when the patient has been upright for some time. This is probably the most common cause of “functional” proteinuria; some reports indicate that it occurs in 15%-20% of healthy young men who have proteinuria on routine urinalysis.)
B. Organic: associated with demonstrable systemic disease or renal pathology.
1. Prerenal proteinuria: not due to primary renal disease.
a. Fever or a variety of toxic conditions. (This is the most common etiology for “organic” proteinuria and is quite frequent.)
b. Venous congestion. (This is most often produced by chronic passive congestion due to heart failure. It is occasionally produced by intraabdominal compression of the renal veins.)
c. Renal hypoxia. Renal hypoxia may be produced by severe dehydration, shock, severe acidosis, acute cardiac decompensation, or severe anemias, all of which lead to a decrease in renal blood flow and sometimes hypoxic renal changes. Very severe hypoxia, especially if acute, may lead to renal tubular necrosis.
d. Hypertension (moderate or severe chronic hypertension, malignant hypertension or eclampsia)
e. Myxedema.
f. Bence Jones protein.
2. Renal proteinuria: primarily kidney disease.
a. Glomerulonephritis.
b. Nephrotic syndrome, primary or secondary.
c. Destructive parenchymal lesions (tumor, infection, infarct).
3. Postrenal proteinuria: protein added to the urine at some point farther down the urinary tract from the renal parenchyma.
a. Infection of the renal pelvis or ureter.
b. Cystitis.
c. Urethritis or prostatitis.
d. Contamination with vaginal secretions. This cause may be suggested by presence of moderate or large numbers of squamous epithelial cells in the urine sediment.
Protein tests are not specific for serum protein. Test results demonstrating protein detect filtered serum protein but may also result from test reaction with WBCs or RBCs, regardless of their origin, if they are present in sufficient number. However, intrinsic renal disease usually (not always) is associated with proteinuria whether abnormal cells are present or not. Thus, by testing the supernate of a centrifuged specimen for protein, one can tentatively assume that WBCs or RBCs originated mainly in the lower urinary tract if pyuria or hematuria occurs in the absence of proteinuria.
Urine protein test methods. There are several clinically acceptable methods for semiquantitative protein testing. The older methods are heat with acetic acid and sulfosalicylic acid (3%, 10%, or 20%). Sulfosalicylic acid (SSA) is slightly more sensitive than the heat-acetic acid method. Sulfosalicylic acid false positive reactions may occur, notably with tolbutamide (Orinase), urates, hemoglobin (RBCs or hemolysis), WBCs, massive penicillin doses, dextran, occasionally with salicylates, and with various radiopaque x-ray substances. Bence Jones protein also gives a positive reaction. Results are expressed as 1+ to 4+; this correlates very roughly with the amount of protein present (Table 12-2).
Table 12-2 Relationship of qualitative and quantitative urine protein results
Kingsbury-Clark procedure. This is a quantitative sulfosalicylic acid method using a series of permanent standards, each representing a different but measured amount of protein. After sulfosalicylic acid is added to the unknown specimen, a precipitate is formed that is compared with the standards and reported as the amount in the particular standard that it matches.
Dipstick methods. Most laboratories now use dipstick methods, usually with multiple tests on the same paper strip. The sulfosalicylic acid method will detect as little as 10 mg/100 ml of protein, whereas the dipsticks are not reliable for less than 20-30 mg/100 ml. The dipsticks will react with hemoglobin but not with the other substances previously listed which might produce false positive results with sulfosalicylic acid. Dipstick behavior with Bence Jones protein is erratic; in many cases the results are positive, but a significant number do not react. Dipsticks are said to give false positive protein results when the urine has a very alkaline pH, although my experience has been that no change occurs even at pH 8.5 (a value that is very rarely exceeded). False negative results may occur if contact of the dipstick with urine is greatly prolonged, since this could wash out the buffer in the protein detection zone on the test strip. In addition, protein readings are only semiquantitative, and it is often difficult to obtain uniform results from the same specimens read by different individuals. This happens because the result depends on a color change corresponding to a change from one quantity (or level) of protein to the next; the color change may not always be clear cut, and interpretation of any color change is always partially subjective. There tends to be a fairly wide variation in results when specimens containing large amounts of protein are tested.
24-hour measurement. Because individuals often vary in their interpretations, and also because the degree of urine concentration may greatly influence results on random urine specimens, it is sometimes useful to determine 24-hour urine protein excretion. A 24-hour specimen may also be affected by degree of concentration but to a lesser extent. However, 24-hour specimens also have their problems. One of these is incomplete specimen collection. Another is variation in results using different test methods or reagents, most of which have relatively poor reproducibility (coefficient of variation exceeding 10%) and react differently with different proteins. In some reports this occasionally led to very substantial differences in results of different methods on the same specimen.
Several studies indicate that the protein/creatinine ratio in single-voided specimens produces an adequate estimation of normality or degree of abnormality compared with 24-hour urine specimens. Since the recumbent and the upright protein excretion rate is often different, first-morning urine specimens are not recommended for this test.
Microalbuminuria. Recently, several studies have suggested that a latent period exists in renal disease associated with insulin-dependent diabetics. In this phase there is on-going glomerular damage that is manifest by increased albumin excretion but in quantities too small to be detectable by standard laboratory protein tests, either dipstick or usual 24-hour procedures. One current definition of diabetic microalbuminuria is albumin excretion of 20-200 µg/minute (30-300 mg/24 hours) found in at least two of three specimens collected within 6 months. Dipsticks will begin detecting proteinuria slightly above the microalbuminuria range upper limit. Multiple samples are required because diabetics with or without microalbuminuria may have day-to-day variation in albumin excretion as high as 50%. Besides diabetics, nondiabetic persons with hypertension (even mild) have increased incidence of microalbuminuria and dipstick-detectable proteinuria (in one study, the mean protein value for hypertensive person was 3 times that of nonhypertensives); and one report indicates increased incidence of proteinuria in patients with heart failure (see also discussion in Chapter 28).
In summary, not all proteinuria is pathologic; even if so, it may be transient. Often the kidney is only indirectly involved, and occasionally protein may be added to the urine beyond the kidney. Individual interpretations of turbidity tests vary significantly and so do various assay methods for 24-hour urine protein.
Glucose
The most common and important glucosuria occurs in diabetes mellitus. The normal renal threshold is usually about 180 mg/100 ml (literature range 165-200 mg/100 ml) serum glucose level; above that, enough glucose is filtered to exceed the usual tubular transfer maximum for glucose reabsorption, and the surplus remains in the urine. However, individuals vary in their tubular transfer reabsorptive capacities. If the capacity happens to be low, the individual may spill glucose at lower blood levels than the average person (“renal glucosuria”). Certain uncommon conditions may elevate blood glucose levels in nondiabetic persons. A partial list of the more important conditions associated with glucosuria includes the following (discussed more fully in Chapter 28):
1. Glucosuria without hyperglycemia
a. Glucosuria of pregnancy (lactosuria may occur as well as glucosuria)
b. Renal glucosuria
c. Certain inborn errors of metabolism (Fanconi’s syndrome)
d. After certain nephrotoxic chemicals (carbon monoxide, lead, mercuric chloride)
2. Glucosuria with hyperglycemia
a. Diabetes mellitus
b. Alimentary glucosuria (hyperglycemia is very transient)
c. Increased intracranial pressure (tumors, intracerebral hemorrhage, skull fracture)
d. Certain endocrine diseases or hormone-producing tumors (Cushing’s syndrome, pheochromocytoma)
e. Hyperthyroidism (occasionally)
f. Occasionally, transiently, after myocardial infarction
g. After certain types of anesthesia, such as ether
The most commonly used tests for urine glucose are glucose oxidase enzyme paper dipsticks such as Clinistix, Tes-Tape, and the multiple-test strips, all of which are specific for glucose. Another widely used test is Clinitest, which, like the old Benedict’s method (rarely used today), is based on copper sulfate reduction by reducing substances and is therefore not specific for glucose or even sugar. In several reported comparisons of these tests, Tes-Tape proved most sensitive but also gave occasional false positives. Clinistix gave few false positives but sometimes gave equivocal results or missed a few positive results that Tes-Tape detected. The copper reduction tests included about 10% positive results from reducing substances other than glucose. Clinitest is a copper reduction tablet test that seemed to have median sensitivity, whereas Benedict’s method, which is a standard type of chemical procedure, was least sensitive, missing 10%-15% of tests positive by glucose oxidase, but with very few false positives apart from other reducing substances.
Dipstick problems. Although theoretically specific and relatively sensitive, the enzyme papers are not infallible. False positive results have been reported due to hydrogen peroxide and to hypochlorites (found in certain cleaning compounds). These substances give negative copper reduction test results. Large amounts of vitamin C may produce false negative results with the glucose oxidase methods but false positive results with reducing substance methods. False negative results using Clinistix (but not Tes-Tape) have been reported from homogentisic acid (alkaptonuria), levodopa, and large doses of aspirin. Also, the enzyme papers may unaccountably miss an occasional positive result that is detected by copper sulfate reduction techniques. As with urine protein dipsticks, studies have demonstrated considerable variation in test reports by different persons on the same specimens, especially at glucose levels that are borderline between two test strip color change levels.
Clinitest (copper reduction) false positive results are produced by sugars other than glucose (galactose, lactose) and by hemogentisic acid (alkaptonuria). Other potentially troublesome substances are p-aminosalicylic acid (PAS), methyldopa (Aldomet), heavy salicylate therapy (salicyluric acid excreted), and heavy concentrations of urates. Various beta-lactam antibiotics in large doses (especially the cephalosporins, but also the penicillins, monobactams, and carbapenems) may interfere with Clinitest interpretation by forming various colors. An important technical consideration is to keep Clinitest tablets and the enzyme papers free from moisture before use.
Change in Clinitest methodology. The original methodology for Clinitest used five drops of urine. When urine contains large amounts of sugar, the color change typical of a high concentration may be unstable and quickly change to another color, which could be misinterpreted as an end point produced by smaller amounts of sugar (“pass-through” phenomenon). Many laboratories now use a two-drop specimen, which avoids the pass-through effect. However, the color changes that represent different concentrations of reducing substances are not the same with five drops as with two drops. Knowledge of the method used is essential for correct interpretation by the patient or the physician.
Acetone and diacetic acid (ketone bodies)
Ketone bodies include b-hydroxybutyric acid (BHBA), diacetic (acetoacetic) acid (DAA), and acetone. Under normal conditions, anaerobic metabolism of glucose proceeds to eventual formation of a compound called acetyl–coenzyme A, or acetyl-CoA. It can also be produced by metabolism of fatty acids. Most acetyl-CoA is used in the tricarboxylic (citric acid) metabolic cycle. However, acetyl-CoA can also enter the pathway to ketone body formation in the liver. Diacetic acid is formed initially, with the great majority being metabolized to BHBA and a small amount spontaneously decarboxylated to acetone and carbon dioxide. The major (but not the only) impetus toward increased ketone formation is either carbohydrate restriction or impaired carbohydrate metabolism. Skeletal and cardiac muscle can utilize a limited amount of DAA and BHBA as an energy source when normal glucose-derived energy is insufficient; but after a certain point, metabolic capacity is exceeded and excess ketone bodies are excreted by the kidney. However, renal excretion rate is also limited, and when ketone formation exceeds muscle utilization and renal excretory capacity, ketone bodies accumulate in the plasma, a condition known as ketosis. Normal plasma ketone composition is about 78% BHBA, 20% DAA, and 2% acetone.
Technical methods and their drawbacks. Most chemical tests for ketones employ a nitroprusside reagent. Nitroprusside detects both DAA and acetone but not BHAA. The most commonly used product of this type is a tablet reagent formulation called Acetest. Acetest will detect concentrations of 5 mg/100 ml of DAA as well as react with acetone and can be used with serum, plasma, or urine. In addition, there is a dipstick method called Ketostix that is specific for DAA and detects 10 mg/100 ml (980 µmol/L). This method is also incorporated into certain multiple-test urine dipsticks from several manufacturers. Dipstick false positive results have been reported with levodopa, mesna, Captopril, and N-acetylcysteine. Since the concentration of DAA is several times that of acetone, and also because Acetest is only about one fifth as sensitive to acetone as it is to DAA, the dipstick tests that detect only DAA give equivalent results in urine to those of Acetest. However, the majority of literature references prefer Acetest when testing plasma or serum. The majority also recommend crushing the Acetest tablet for serum but not for urine.
Some case reports describe instances in which the dipstick test for ketones failed to detect ketones in the plasma of certain patients with diabetic ketoacidosis. Acetest tablets were able to detect the ketones. The manufacturer states that the ketone portion of multiple-test strip dipsticks is more sensitive to effects of moisture than the other test areas and that the dipstick container lid must be replaced and fastened immediately after a test strip is removed (which is an unrealistic expectation in many laboratories). Free sulfhydride compounds (such as N-acetylcysteine used to treat acetaminophen overdose) will produce false positive reaction for ketones in tests based on nitroprusside. False positive reactions due to sulfhydride can be detected by adding glacial acetic acid to the reagent area after the reaction and seeing the reaction fade completely by 30 seconds or less.
Interpretation of test results. Standard chemical tests for ketone bodies do not detect the small amounts in normal plasma or urine. A positive test result in both urine and plasma is uncommon and usually indicates severe excess of ketones. This is most often due to severe metabolic acidosis, of which the classic example is diabetic acidosis. Detectable urine ketones (ketonuria) with nondetectable plasma (or serum) ketones is fairly common and indicates mild or moderate over-production of ketone bodies. In one study, it was reported in 14%-28% of all hospital admission urines. Ketonuria is a classic finding in diabetic acidosis. However, ketonuria may also be found in starvation, in alcoholism, in many normal pregnancies at time of delivery, and especially in severe dehydration from many causes (vomiting, diarrhea, severe infections). In children, ketone bodies are prone to appear on what, by adult standards, would be relatively small provocation. A weakly positive urine test result for ketones is usually of little significance.
It must be emphasized that diabetes is practically the only disease in which ketonuria has real diagnostic importance. Slight to moderate degrees of ketonuria are fairly common in certain other conditions, as mentioned earlier, but are only incidental. Serum ketone results are usually negative. In my experience, trace, 1+, and 2+ (using a scale of 0-4+) urine ketone results are rarely accompanied by positive serum ketone results, and the same was true even for some patients with 3+ ketonuria. In diabetes the presence of large amounts of urine ketones raises the question of a possible dangerous degree of diabetic ketoacidosis. In well-established diabetic ketoacidosis the serum or plasma acetone test result will usually be positive (exceptions are the rare cases of hyperosmolar coma or lactic acidosis). In fact, the quantity of plasma ketones, estimated by testing dilutions of plasma, provides some (although not an exact) indication of the severity of acidosis and the amount of insulin needed. In the urine, nitroprusside test results usually will be strongly positive. In most cases, there will be glucosuria as well as marked ketonuria. The major exception is renal insufficiency with oliguria due to severe renal disease, shock, or exceptionally severe acidosis with secondary renal shutdown, since some of these patients may be unable to filter the increased serum ketone bodies into the urine. The blood urea nitrogen (BUN) level is frequently elevated in diabetic acidosis; even so, there usually will be ketonuria. If there is any question, a plasma or serum ketone test should be done.
In summary, the urine ketone result in severe diabetic acidosis usually is very strongly positive and the plasma ketone result is positive. However, if symptoms of diabetic acidosis are present, lack of strongly positive urine ketone results does not rule out the diagnosis if renal failure is present.
Serum ketone quantitative measurement. Biochemical methods (usually enzymatic) are available to measure BHBA, which actually is the dominant ketone form in ketosis and provides a better estimation of severity in diabetic acidosis than DAA measurement. In early ketoacidosis, the BHBA level becomes elevated, whereas the DAA level is still normal. However, BHBA has not replaced DAA because of the ease, convenience, speed, and low cost of Acetest and the dipsticks.
Hemoglobin (blood)
Characteristics of test methods. Several biochemical procedures for detecting hemoglobin in urine are on the market. Some have a sensitivity of approximately 1:100,000, about that of the old benzidine method. A representative of this type is an orthotolidine reagent tablet method called Occultest. This is reported to detect fewer than five RBCs per high-power field in centrifuged urine sediment. Hematest is another tablet test with the same basic reagents but is adjusted to approximately 1:20,000 sensitivity, slightly more than the guaiac method. Experimentally, it will not reliably detect fewer than 200 RBCs per high-power field unless some of the cells have hemolyzed. It is much more sensitive to free hemoglobin, where it can detect amounts produced by hemolysis of only 25-30 RBCs per high-power field. It has been used for feces as well as urine, although at this sensitivity there is an appreciable number of false positives. A dipstick called Hemastix and the occult blood section of different multitest dipsticks are likewise based on orthotolidine but are more sensitive than the reagent tablets, detecting 0.04 mg/100 ml of hemoglobin, or, in my experience, more than two to three RBCs per high-power field of centrifuged urine sediment. There is a small but probably significant variation in sensitivity among the different dipsticks. The usefulness of these chemical tests is enhanced by their ability to detect hemoglobin even after some or all of the RBCs have disintegrated and are no longer visible microscopically. Red blood cell lysis is particularly likely to occur in alkaline or dilute urine. Failure to mix a specimen before testing could result in testing a supernate without RBCs and could yield a false negative dipstick reaction. Large doses of vitamin C interfere with the test chemical reaction and may produce a false negative result. One report indicates that providone-iodine (Betadine) can produce a false positive reaction. Contamination from vaginal secretions in the female may introduce RBCs into the specimen as an artifact. Myoglobin reacts as well as hemoglobin.
Biochemical methods versus microscopic examination. There is disagreement in the literature as to whether the dipstick tests for hemoglobin could be used in place of microscopic examination (for RBCs) of the urine sediment. In my experience with one company’s multitest dipstick, almost all patients with microscopic findings of two or more RBCs per high-power field were detected by the dipstick. However, testing with dipstick only would not reveal abnormal numbers of squamous epithelial cells, which would raise the question of possible contamination artifact. Also, detection of urine hemoglobin or hematuria would necessitate a microscopic sediment examination for RBC casts. Etiologies of hematuria are discussed later in the section on microscopic sediment examination.
Leukocyte esterase (white blood cells)
A dipstick called Chemstrip-L that will detect WBCs in urine by means of hydrolysis of the reagent by esterase enzyme contained in the cytoplasm of neutrophils has been introduced. The same reagent has been incorporated into some of the multitest dipsticks. Reports indicate that the leukocyte esterase test will detect about 85%- 95% (range, 73%-99%) of patients with abnormal numbers of WBCs in centrifuged urinary sediment. The test reportedly will also detect abnormal numbers of WBCs that have lysed due to alkaline urine or other factors. The leukocyte esterase test is about 80%-90% sensitive in detecting urinary tract infection (defined as 100,000 colony-forming units/ml) compared with urine culture. When performed with the nitrite test (discussed later), sensitivity versus that of quantitative urine culture improves to about 85%-90% (range, 78%-100%). Several reports suggest false positive results due to Trichomonas organisms, but others suggest relatively little interference. In addition, leukocyte esterase will not differentiate urinary tract WBCs from WBCs added through contamination of the specimen (especially by vaginal secretions) during specimen collection.
Nitrite
Many bacteria produce an enzyme called reductase that can reduce urinary nitrates to nitrites. Some of the multitest dipsticks contain a reagent area that reacts with nitrite to produce a color, thus indirectly suggesting the presence of bacteria in certain quantity. Sensitivity of the nitrite test versus that of quantitative urine culture is only about 50% (range, 35%-69%). However, it can enhance the sensitivity of the leukocyte esterase test to detect urinary tract infection.
Bile (conjugated bilirubin)
Conjugated bilirubin is not normally detected in urine but can appear secondary to biliary tract obstruction, either extrahepatic (common duct obstruction) or intrahepatic (cholestatic liver cell injury due to such conditions as active cirrhosis or hepatitis virus hepatitis). Typically, in hepatitis virus hepatitis the urine appears dark 2 or 3 days before the patient becomes icteric and clears swiftly once skin jaundice develops.
In pulmonary infarction, some elevation in the serum unconjugated bilirubin level may occur, but unconjugated bilirubin does not appear in urine.
Tests for conjugated bilirubin in urine include Fouchet’s reagent, Ictotest tablets, or dipstick tests. The simple foam test (yellow foam after shaking) may be all that is necessary (false positive result may be produced by pyridium). Ictotest and the dipstick tests are based on the same chemical reaction and are fairly sensitive, detecting as little as 0.1 mg/100 ml (7.1 µmol/L). According to one report, the dipsticks detect about 70% of patients with serum conjugated bilirubin between 1.0-2.0 mg/100ml (17.1-34.2 µmol/L) and nearly all above 2.0 mg/100 ml (34.2 µmol/L). These tests are reasonably specific, but chlorpromazine may produce false positive results. If urine is allowed to stand for some time before testing, conjugated bilirubin decomposes and a false negative result may occur. Ictotest is somewhat less sensitive to this change than the dipsticks and therefore is a little less likely to become falsely negative.
Most requests for urine bile could be questioned, since the serum bilirubin level would provide more useful information (Chapter 20).
Urobilinogen
Urobilinogen is produced from conjugated bilirubin by metabolic activity of bacteria in the intestine, followed by reabsorption into the bloodstream (Chapter 20). Urine urobilinogen becomes increased from either a marked increase in production secondary to increase in serum unconjugated bilirubin (usually from hemolytic processes) or because of inability of damaged liver parenchymal cells to metabolize normal amounts of urobilinogen absorbed from the intestine (usually due to cirrhosis or severe hepatitis). The metabolic pathways involved are discussed in the chapter on liver function tests.
Tests for the presence of urobilinogen include both a standard chemistry method and a dipstick procedure, both based on Ehrlich’s reagent. Either a random urine specimen or a 24-hour specimen can be used. The 24-hour specimen must contain a preservative.
For routine specimens, it is of great importance that the test be done within 30 minutes after voiding. Urobilinogen rapidly oxidizes in air to nondetectable urobilin, and without special preservative it will decrease significantly after 30 minutes. This unrecognized fact probably accounts for the test’s poor reputation. If the procedure cannot be done relatively soon after voiding, it is better to get a 24-hour specimen with preservative.
Urine urobilinogen determination is rarely needed, since liver function or other tests provide more useful information.
Urine urobilinogen methods based on Ehrlich’s reagent may also detect porphobilinogen; methods using other reagents will not.
Microscopic examination of urinary sediment
Urine microscopic examination is routinely done on centrifuged urinary sediment. There is no widely accepted standardized procedure for this, and the varying degrees of sediment concentration that are produced make difficult any unquestioning interpretation of quantitative reports. However, it is fairly safe to say that normally so few cellular elements are excreted that most results of examination in normal uncontaminated specimens fall within normal clinical values no matter how much the specimen is centrifuged. The majority of sources (including my experience) use a reference range of 0-2 RBCs or 0-5 WBCs per high-power field and only an occasional cast. However, there is disagreement in the literature regarding the reference range for both WBCs and RBCs. Reference ranges for WBC can be found that vary from 1 WBC per high-power field to as many as 20 WBCs per high-power field. RBC ranges are discussed in the section on hematuria investigation in Chapter 13. Some commercial systems are available for preparation of the urine sediment that can improve reproducibility.
The main pathologic elements of urinary sediment include RBCs, WBCs, and casts. Less important elements are yeasts, crystals, or epithelial cells.
Red blood cells. Gross urinary bleeding is usually associated with stones, acute glomerulonephritis, and tumor, although these conditions may (less often) be manifested only by microscopic hematuria. Tuberculosis traditionally has been listed as a fourth cause but is rare today. In several series, benign prostate hyperplasia and bladder or urethral infection were also frequent etiologies. There are conditions that frequently are associated with significant microscopic hematuria and occasionally approach gross bleeding. Some of the more important include bleeding and clotting disorders (e.g., purpura or anticoagulants), blood dyscrasias (including sickle cell anemia or leukemia), renal infarction, malignant hypertension, subacute bacterial endocarditis, collagen diseases (especially lupus and polyarteritis nodosa), Weil’s disease, and certain lower urinary tract conditions such as cystitis, urethritis, and prostatitis. In the female, vaginal blood or leukocytes may contaminate ordinary voided specimens; finding significant numbers of squamous epithelial cells in the urinary sediment suggests such contamination. Yeast may simulate RBCs (discussed later).
Red blood cell casts are the most reliable way to localize the source of bleeding to the kidney. Some reports indicate that the presence of “dysmorphic RBCs” (distorted or shrunken RBCs) using ordinary microscopy, phase contrast, Wright’s stain on a sediment smear, or a Papanicolaou cytology technique is also helpful in suggesting renal origin.
White blood cells. WBCs may originate anywhere in the urinary tract. Hematogenous spread of infection to the kidney usually first localizes in the renal cortex. Isolated cortical lesions may be relatively silent. Retrograde invasion from the bladder tends to involve calyces and medulla initially. Urinary tract obstruction is often a factor in retrograde pyelonephritis. Generally speaking, pyuria of renal origin is usually accompanied by significant proteinuria. Pyuria originating in the lower urinary tract may be associated with proteinuria, but it tends to be relatively slight.
Urinary tract infections tend to be accompanied by bacteriuria. Tuberculosis of the kidney, besides producing hematuria, characteristically is associated with pyuria without bacteriuria. An ordinary urine culture would not reveal tuberculosis organisms. WBC casts are definite evidence that urinary WBCs originate in the kidney (discussed later); WBCs in clumps are suggestive of renal origin but are not diagnostic.
Correlation of pyuria with urine culture. Although many physicians regard pyuria as a good screening test for urinary tract infection, false normal results are reported in approximately 25%-30% of patients (literature range 0%-87%) who have a positive quantitative urine culture result. WBC counting of uncentrifuged urine in a WBC counting chamber is reported to be more accurate than urine sediment, but very few laboratories use this technique. Abnormal numbers of WBCs are a reasonably good indicator of urinary tract infection, although reports indicate 2%-13% false positive results compared to urine culture. In females, one should take precautions to avoid artifactual increases in urine WBCs from contamination by vaginal or labial secretions. Several studies (including my own experience) have suggested female urine contamination rates as high as 30%. In any specimen it is important to perform the microscopic examination promptly (i.e., within 1 hour after voiding) or else use some method of preservation. WBCs lyse in hypotonic or alkaline urine. Some studies reported that as many as 30%-50% of specimens containing abnormal numbers of WBCs were normal after 2-3 hours of standing at room temperature.
Casts. Casts are protein conglomerates that outline the shape of the renal tubules in which they were formed. Factors involved in cast formation include the following:
1. pH: Protein casts tend to dissolve in alkaline medium.
2. Concentration: Casts tend to dissolve in considerably dilute medium. Concentration also has an important role in the formation of casts; a concentrated urine favors precipitation of protein.
3. Proteinuria: Protein is necessary for cast formation, and significant cylindruria (cast excretion) is most often accompanied by proteinuria. Proteinuria may be of varied etiology. The postrenal type is added beyond the kidneys and obviously cannot be involved in cast formation.
4. Stasis: Stasis is usually secondary to intratubular obstruction and thus allows time for protein precipitation within tubules.
Mechanisms of cast formation. Mechanisms of cast formation are better understood if one considers the normal physiology of urine formation. The substance filtered at the glomerulus is essentially an ultrafiltrate of plasma. In the proximal tubules, up to 85% of filtered sodium and chloride is reabsorbed, with water passively accompanying these ions. In the thick ascending loop of Henle, sodium is actively reabsorbed; however, since the membrane here is impermeable to water, an excess of water (free water) remains. As water passes along the distal tubules, some may be reabsorbed with sodium ions, and in the collecting tubules, up to 5% of the glomerular filtrate is osmotically reabsorbed due to the relatively high osmolality of the renal interstitial cells. Water reabsorption in distal and collecting tubules is under the control of antidiuretic hormone. At this point, the urine reaches its maximum concentration and proceeds to the bladder relatively unchanged. Thus, cast formation takes place ordinarily in the distal and collecting tubules, where acidification takes place and concentration reaches its height.
Cast types. There are two main types of casts, cellular and hyaline, depending on their main constituents. Other types include fatty casts, broad casts, and hemoglobin casts.
CELLULAR CASTS. RBCs, WBCs, desquamated renal epithelial cells, or any combination of these may be trapped inside renal tubules in a protein matrix. The cast is named for the cells inside it. This proves renal orgin of the RBCs and WBCs. (Fig. 12-1).
Fig. 12-1 Formation of casts.
As the cellular cast moves slowly down the nephron, the cells begin to disintegrate. Eventually, all that is left of the cells are relatively large fragments, chunks, or granules. The cellular cast has now become a coarsely granular cast. It is composed entirely of large irregular or coarse solid granules.
If disintegration is allowed to continue, the coarse granules break down to small granules, and a relatively homogeneous finely granular cast is formed.
The end stage of this process is the production of a homogeneous refractile material still in the shape of the tubule, known as a waxy cast. The waxy cast is translucent, without granules, and reflects light, which gives it a somewhat shiny, semisolid appearance. What stage of cast finally reaches the bladder depends on how long the cast takes to traverse the nephron and thus how long it remains in the kidney, where the forces of disintegration may work. A waxy cast thus indicates fairly severe stasis in the renal tubules.
HYALINE CASTS. Hyaline casts are composed almost exclusively of protein alone, and they pass almost unchanged down the urinary tract. They are dull, nearly transparent, and reflect light poorly compared with waxy casts. Thus, hyaline casts are often hard to see, and the microscope condenser usually must be turned down to give better contrast.
Sometimes cellular elements may be trapped within hyaline casts. If hyaline material still predominates, the result is considered a hyaline cast with cellular inclusions. Degenerative changes can take place similar to those of regular cellular casts, with the production of hyaline coarsely granular and hyaline finely granular casts.
FATTY CASTS. Fatty casts are a special type of cellular cast. In certain types of renal tubular damage, fatty degeneration of the tubular epithelial cells takes place. These cells desquamate and are incorporated into casts. The epithelial cells contain fatty droplets. As the cells degenerate, the fatty droplets remain and may even coalesce somewhat. The final result is a cast composed mainly of fatty droplets and protein. Sometimes either renal epithelial cells with fatty degeneration or the remnants thereof containing fat droplets are found floating free in the urine and are called oval fat bodies.
When oval fat bodies or fatty casts are present in significant number, the patient usually has a disease that is associated with the nephrotic syndrome, such as primary lipoid nephrosis or nephrosis secondary to Kimmelstiel-Wilson syndrome, systemic lupus, amyloid, subacute glomerulonephritis, the nephrotic stage of chronic glomerulonephritis, certain tubule poisons such as mercury, and rare hypersensitivity reactions such as those from insect bites. Oval fat bodies have essentially the same significance as fatty casts. Fat droplets have a Maltese cross appearance in polarized light but are recognized without polarized light. They can also be identified using fat stains such as Sudan IV.
BROAD CASTS. When very severe stasis occurs regardless of the type of cast involved, cast formation may take place in the larger more distal collecting tubules, where large ducts drain several smaller collecting tubules. If this occurs, a broad cast is formed, several times wider than ordinary-sized casts. This is always indicative of severe localized tubule damage. These broad casts may be of any cast type, and due to their peculiar mode of formation they are sometimes spoken of as “renal failure casts.” This is not entirely accurate, because the kidney may often recover from that particular episode and the problem sometimes involves only part of a kidney.
HEMOGLOBIN CASTS. Hemoglobin casts are derived from RBC casts that degenerate into granular (rarely, even waxy) material but still have the peculiar orange-red color of hemoglobin. Not all casts derived from RBCs retain this color. Strongly acid urine changes the characteristic color of hemoglobin to the nonspecific gray-brown color of acid hematin. Also, one must differentiate the brown or yellow-brown coloring derived from bile or other urine pigments from the typical color of hemoglobin.
Significance. The significance of casts in the urine varies according to the type of cast. Fatty casts, RBC casts, and WBC casts are always significant. The only general statement one can make about ordinary hyaline or granular casts is that their appearance in significant numbers has some correlation with the factors involved in cast formation—proteinuria, concentration, and stasis. Of these, the most general correlation is with stasis, although one cannot tell whether the stasis is generalized or merely localized. It is common for showers of casts to clear dramatically once the underlying cause of their formation is corrected. On the other hand, significant numbers of casts that persist for some time despite therapeutic efforts may suggest serious intrarenal derangement. In this respect, granular casts in the late stages of development will be seen. Generally speaking, hyaline casts alone are of little practical importance and are usually only an acute, mild, and temporary phenomenon. Thus, to have any meaning, the appearance of casts must be correlated with other findings and with the clinical situation. A few hyaline or granular casts in themselves have no importance.
Crystals. Crystals are often overemphasized in importance. They may, however, be a clue to calculus formation and certain metabolic diseases. Crystals tend to be pH dependent:
Acid urine: uric acid, cystine, calcium oxalate.
Alkaline urine: phosphates, as triple phosphate (magnesium ammonium phosphate), which comprises many staghorn calculi. Note: alkaline urine is most often produced by (Proteus) infection.
Amorphous (loose finely granular) crystals: If the pH is acid, the material is urate; if the pH is alkaline, it is phosphate. Rarely one might find sulfa crystals in acid urine, since sulfadiazine is still sometimes used. Most sulfa crystals resemble needle-like structures in bunches, but this appearance can be mimicked by certain nonpathologic crystals.
Epithelial cells. Squamous epithelial cells are useful as an index of possible contamination by vaginal secretions in females or by foreskin in uncircumcised males. I have found (unpublished study) that more than 10 squamous epithelial cells per low-power (10 Ч objective) field provide an adequate guidepost for suspicion of contamination. However, contamination (especially bacteriologic contamination) might occur without abnormal numbers of squamous cells, and the presence of abnormal numbers of squamous cells does not mean that RBCs and WBCs are present only because of contamination. The squamous cells only alert one to a definite possibility of contamination and, if abnormalities are present, suggest the desirability of a carefully collected midstream specimen. Unfortunately, however, even specimens that supposedly are collected by careful midstream voiding technique may be contaminated, especially if the patient collects the specimen without expert assistance.
Miscellaneous microscopic sediment findings.
Trichomonas. Female vaginal infection by the protozoan parasite Trichomonas is fairly common; it is often associated with mild proteinuria, WBCs, and epithelial cells in the urine sediment. The organism is frequently found in urine specimens, where diagnosis is greatly assisted by the organism’s motility (so a fresh specimen is essential). When nonmotile, it resembles a large WBC or small tubular epithelial cell. Fluorescent antibody methods are now available for diagnosis and are more sensitive than ordinary urine sediment examination but are relatively expensive. Trichomonas is found occasionally in the male.
Spermatozoa. Spermatozoa may appear in the urine of males and, occasionally, in that of females.
Yeast. Yeast is a relatively common sediment finding; the most common is Candida albicans (Monilia). Yeast cells are often misdiagnosed as RBCs. Differential points are budding (not always noticed without careful search), ovoid shape (not always pronounced), an appearance slightly more opaque and homogeneous than that of the RBC, and insolubility in both acid and alkali. If the cells are numerous and their identity is still in doubt, a chemical test for blood is recommended.
Pitfalls in microscopic examination of urine sediment. The most common and most important mistake is failure to mix the specimen sufficiently before a portion of it is poured into a centrifuge tube for concentration of the sediment. Certain other points deserve reemphasis. If the specimen remains at room temperature for a long time, there may be disintegration of cellular elements, bacterial growth, and change toward an alkaline pH. If RBC or WBC casts are not reported, this does not mean that they are not present, since it usually takes careful and somewhat prolonged search to find them (especially RBC casts).
Other urine tests
Calcium. Urine can be tested for calcium with Sulkowitch reagent; it is a semiquantitative method in which results are reported as 0-4+ (normal is 1+) or as negative, moderately positive, and strongly positive. Standard clinical chemistry quantitative methods have almost entirely replaced this test. Interpretation of urine calcium is discussed in Chapter 25. Excessively concentrated or diluted urine may produce unreliable results, just as it does for protein.
Porphobilinogen. Porphobilinogen is diagnostic of acute porphyria (episodes of abdominal crampy pain with vomiting and leukocytosis, Chapter 34). Porphobilinogen is colorless and is easily identified by rapid screening tests, such as the Watson-Schwartz procedure.
Urinary coproporphyrins. Coproporphyrin type III excretion is increased in lead poisoning (Chapter 35) and has been used as a relatively simple screening test for that condition. However, increased coproporphyrin III is not specific for lead poisoning.
Phenylpyruvic acid. Phenylpyruvic acid is excreted in phenylketonuria (Chapter 34). A ferric chloride test is the classic means of detection. There is a dipstick method (Phenistix) for the diagnosis of phenylketonuria that depends on the reaction between ferric ions and phenylpyruvic acid, as does the classic ferric chloride test. It is more sensitive than ferric chloride, detecting as little as 8 mg/100 ml or trace amounts of phenylpyruvic acid in urine. False positive results occur if large amounts of ketone bodies are present. Reactions will take place with salicylates, PAS, and phenothiazine metabolites, but the color is said to be different from that given by phenylpyruvic acid. The urine screening tests have become less important since neonatal blood test screening has become mandatory in many areas. It takes approximately 3-6 weeks after birth before phenylpyruvic acid becomes detectable in urine, and by that time treatment is much less effective.
Points to remember in interpretation of urinalysis results
1. Laboratory reports usually err in omission rather than commission (except occasionally in RBCs vs. yeast). If technicians cannot identify something, they usually do not mention it at all.
2. Certain diagnostic findings, such as RBC casts, may not appear in every high-power (or even every low-power) field. If hematuria is present, one should look for RBC casts and, similarly, for WBC casts and other such structures in the appropriate sediment settings.
3. If laboratory personnel know what the physician is trying to find, they usually will give more than a routine glance. Otherwise, the pressure of routine work may result in examination that is not sufficient to detect important abnormalities.
4. In many laboratories, reports will include only items specifically requested, even when other abnormalities are grossly visible (e.g., bile).
5. Contamination of urine by vaginal secretions in the female may introduce RBCs, WBCs, or protein into the specimen as an artifact. The presence of more than 10 squamous epithelial cells per low-power field is a warning of possible contamination. Nevertheless, abnormalities should not be dismissed on the basis of possible contamination without careful and repeated attempts to obtain a noncontaminated specimen, because contamination may be superimposed on true abnormality. In some cases this problem may necessitate expert help in specimen collection.
One fact that should be stressed when commercial tests of any type are used is to follow all the directions exactly. Any dipstick or table method requires a certain length of time in contact with the specimen. When this is true, a quick dip-and-read technique, or, on the other hand, overly prolonged contact with the specimen, may lead to false results. Moisture may affect some of the test reagents, so the tops on reagent bottles should be replaced and tightened immediately after the dipsticks or tablets are taken out.
Laboratory evaluation of urinary tract calculi. Laboratory evaluation consists of (1) stone mineral analysis, and (2) investigation of stone-formation etiology. Stone mineral analysis is performed by x-ray diffraction, infrared spectroscopy, optical crystallography (polarization microscopy), or some combination of these techniques. Standard chemical analysis of crushed stone material is no longer recommended, due to fragment inaccuracies, interferences, lack of sensitivity, and problems with sufficient specimen if the stone is small. There is no universally accepted standard workup protocol to investigate pathogenesis. However, the majority of investigators require both a 24-hour urine specimen and a fasting serum specimen obtained on the same day, on three separate occasions. The urine specimen should be kept on ice or in the refrigerator during collection. In the laboratory, the specimen is preserved in the refrigerator if necessary tests are performed in house; if the specimen is sent to an outside laboratory, appropriate preservatives should be added. The patient is usually on his or her regular diet (at least initially). The substances measured vary somewhat among investigators, but measurements most often include calcium, phosphate, uric acid, oxalate, urine pH, and urine volumes. Citrate and cystine are also frequently assayed.
Representative reference ranges (24-hour urine) are as follows: calcium, <250 mg (6.24 mmol)/day on normal diet and <150 mg/(6.24 mmol)/day on low-calcium diet; uric acid, 250-800 mg (1.49-4.76 mmol)/day on normal diet and <600 mg (3.57 mmol)/day on low-purine diet; phosphate, 400-1,300 mg (12.9-42 mmol)/day; and oxalate, 10-40 mg (0.11-0.44 mmol)/day.