Why is a first voided morning specimen preferred for performing a microscopic examination of urine?

Casts

Casts are cylindrical structures that form in the lumen of distal renal tubules and collecting ducts. Their matrix is made of Tamm-Horsfall glycoprotein, today known as uromodulin, which is secreted by the cells of the thick ascending limb of Henle loop. Trapping of particles within the cast matrix results in casts with different appearances, each of which may have specific clinical significance (Table 4.2). Because casts form in the renal tubules, whatever particle is contained in a cast derives from the kidneys. Specific casts include the following:

Hyaline casts are colorless with a low refractive index (seeFig. 4.3A). They are easily seen with phase contrast microscopy but can be overlooked when bright-field microscopy is used. Hyaline casts may occur in normal urine, especially when it is concentrated and acidic (both conditions favor precipitation of uromodulin). In patients with renal disease, hyaline casts are usually associated with other types of casts.

Hyaline-granular casts contain variable amounts of granules within the hyaline matrix (seeFig. 4.3B) and are the most common mixed casts (see later discussion). Hyaline-granular casts are rare in normal individuals but are common in patients with renal diseases such as GN28 and acute interstitial nephritis.32

Granular casts can be finely granular (seeFig. 4.3C) or coarsely granular. Both types indicate renal disease. In patients with AKI, granular casts together with RTECs30 or with epithelial casts33 are a sensitive marker of ATN.

Waxy casts derive their name from their appearance, which is similar to that of melted wax (seeFig. 4.3D). They are typically found in patients with renal disease associated with impaired renal function, whether acute, rapidly progressive, or chronic.34

Fatty casts contain variable amounts of lipid droplets, isolated, in clumps, or packed or even oval fat bodies or cholesterol crystals. Fatty casts are typical of glomerular diseases associated with marked proteinuria or the nephrotic syndrome.

Erythrocyte casts may contain a few erythrocytes (seeFig. 4.3E) or so many that the matrix of the cast cannot be identified. Erythrocyte casts are usually considered a marker of glomerular bleeding, although a recent report found them in 28% of patients with acute interstitial nephritis.32

Hemoglobin casts generally have a brownish hue and a coarsely granular appearance, which derives from the degradation of erythrocytes entrapped within the cast matrix (seeFig. 4.3F). In such cases, hemoglobin casts have the same clinical significance as erythrocyte casts. However, hemoglobin casts also may derive from hemoglobinuria, as may occur in intravascular hemolysis. In these patients, hemoglobin casts have a smooth surface.

Leukocyte casts contain variable amounts of polymorphonuclear leukocytes (seeFig. 4.3G). They can be found in patients with acute pyelonephritis and acute interstitial nephritis, as well as in active proliferative GN.28

Renal tubular epithelial cell casts (so-called epithelial casts) contain variable numbers of RTECs, which can be identified by their prominent nucleus (seeFig. 4.3H). Epithelial casts indicate damage of the renal tubular epithelium and can therefore be found in the urine of patients with ATN,30 acute interstitial nephritis, and glomerular disease.28

Myoglobin casts are pigmented cylinders, with the myoglobin providing their color. They may be similar to hemoglobin casts (seeFig. 4.3F), from which they can be distinguished by the clinical setting. Myoglobin casts are observed in the urine of patients with AKI associated with rhabdomyolysis.

Bilirubin casts are cylinders pigmented with bilirubin, which can stain any particle contained in the cast (seeFig. 4.3I). They are observed in the urine of patients with jaundice associated with increased direct (conjugated) bilirubin.

Casts containing microorganisms (bacteria and yeasts) indicate renal infection.

Casts containing crystals indicate that crystals derive from the renal tubules. Crystal casts are an important diagnostic element in crystalline-induced nephropathies, such as acute urate nephropathy.35

Mixed casts contain components of different nature, such as granules, cells, and lipids. This causes the appearance of pleomorphic cylinders, whose clinical significance is the same as that for the pure types of casts, of which mixed casts contain some components.

Urine Collection

The way urine is collected and handled can greatly influence the results (Fig. 4.1). Written instructions should be given to the patient as to how to perform a urine collection.3 First, strenuous physical exercise (e.g., running, soccer match) must be avoided in the 72 hours preceding 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. The first or second morning urine specimen is recommended.3

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.3 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. Urine can also be collected through a bladder catheter, although the catheter may cause hematuria. Permanent indwelling catheters are commonly 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.3 The label should identify the patient as well as the hour of urine collection.

Several elements (but especially leukocytes) can lyse rapidly after collection, and the best preservation method to minimize this is uncertain. Refrigeration of specimens at +2°C to +8°C assists preservation but may allow precipitation of phosphates or uric acids, which can hamper examination of the sample. Formaldehyde, glutaraldehyde, CellFIX (a formaldehyde-based fixative),4 and tubes containing a lyophilized borate-formate-sorbitol powder5 are good preservatives for the formed elements of urine.

Urinalysis and Urine Microscopy

In the setting of AKI, a dipstick urinalysis may reveal microhematuria or proteinuria but must be interpreted in conjunction with more specific tests such as a ratio of spot urinary protein or albumin to creatinine and urine microscopy. Dipstick urinalysis may not detect immunoglobulin free light chains (FLCs) or may be falsely positive for protein in the setting of radiographic contrast or alkaline urine. In conjunction with urine microscopy, the presence of urinary hemoglobin or myoglobin by dipstick can be further differentiated. Urine microscopy has been validated as a diagnostic and prognostic tool in hospitalized patients with AKI.33 A fresh urine sample is centrifuged, and the sediment is examined for the presence of cells, casts, and crystals (Figs. 68.4 and68.5) (see alsoChapter 4). Urine microscopy in early prerenal AKI is typically normal with occasional hyaline casts. “Muddy brown” granular casts and renal tubular epithelial cells in the urine support ATN-related AKI. In hospitalized patients with AKI, the presence of more than 10 granular casts per low-power field had a positive predictive value of 100% for a final diagnosis of ATN and a urine sediment score based on granular casts and renal tubular epithelial cells was directly associated with worsening AKI. Thus urine microscopy is useful both in distinguishing ATN-related AKI from prerenal AKI and in predicting the severity of AKI.33

Findings on urinalysis and urine microscopy (seeChapter 4) may suggest CKD (broad, waxy casts) but, more important, may be diagnostic clues to a rare cause of AKI. Proliferative GN may be characterized by considerable microhematuria and proteinuria by dipstick and an active urinary sediment consisting of red blood cells (RBCs) and RBC casts. In this setting the history and physical examination findings should be supported by serologic testing and a kidney biopsy, if the kidneys are normal in size. The presence of white blood cells (WBCs) in clumps and casts, in the absence of bacteria, suggests AIN.26 Renal tubular epithelial cells, granular casts, RBCs, and, rarely, even RBC casts can occur in patients with AIN, whereas urinary eosinophils are no longer considered helpful in diagnosing AIN (seeChapter 60).40 A urine sediment with abundant uric acid crystals accompanying high serum phosphorus levels in a patient undergoing chemotherapy may indicate tumor lysis syndrome (TLS).

Urinalysis as validation of drug use self-report

Urinalysis is a method widely used by clinicians and researchers alike to validate self-report of substance use by youth. While urinalysis has some utility as a validity criterion, particularly for THC (i.e., marijuana and hashish), research findings generally suggest a low association between substance use self-report and drug test findings (McLaney, Del-Boca & Babor, 1994). Interestingly, this is not due to low rates of admitting to drug use, but rather to the discordant pattern of negative drug screens in the face of self-reports of drug use. Drug test results are influenced by several factors, including the amount and frequency of drug use. Additional factors include drug test sensitivity and the amount of time between use and sample collection. Moreover, false positive conclusions (i.e., when screen results are positive yet screened substance was not used) may result from the use of common diet pills and decongestants that mimic, for example, opiate use. Conversely, there are concerns that false-negative readings (i.e., screen results are negative yet the substance used) can be produced by the youth using diuretics, diluting the sample, or adding large quantities of salt to the sample.

What interpretations can be inferred from urinalysis? A positive result on a urinalysis indicates that the identified drug was in the adolescent’s urine. A positive result, however, does not provide any specific information about the client’s use, including how much the indicated substance was used or the pattern or method of use. In addition, a positive finding alone does not suggest abuse or dependence. Thus, incorporating random drug screens during treatment or research is only suggestive of the adolescent’s pattern of use. Three plausible conclusions have been suggested when a drug screen is negative (Manno, 1986). First, the adolescent may not be using drugs. Second, the urinalysis may not be sensitive enough to detect the drug in question. The best example of this problem in relation to drug testing among youth is the length of time a urinalysis can successfully detect alcohol use. Specifically, alcohol generally can only be detected if consumed within six to ten hours prior to when the urine sample is taken. Thus, urinalysis could reveal a negative finding for youth who use alcohol on a near-daily basis. The length of the urinalysis detection period is fairly limited across a variety of drugs, including eight hours for LSD, between one and two days for amphetamines and heroin, two to four days for cocaine, and two to ten days for barbiturates. Urinalysis is better at detecting THC (marijuana and hashish) and benzodiazepines for periods up to six weeks after use. Finally, a negative drug test finding may also result if a given drug is used too little or too infrequently or if the adolescent has tampered with the sample.

Although research examining the utility of drug testing is needed, perhaps the greatest advantage of the drug test is not so much in its ability to accurately indicate an adolescent’s substance use, but rather in the message it conveys to a prospective client or study participant that a method is being employed to check the honesty of their answers. This message could possibly alienate the adolescent by suggesting mistrust or just as easily promote honesty by virtue of the adolescent’s assumption that the drug test will reveal the truth.

I Urinalysis1:Table 19.1

A.

Common indications include: Infectious workup (urinary tract infection [UTI], pyelonephritis), abdominal trauma, suspected diabetes or renal disease, rhabdomyolysis, edema, failure to thrive.

Best if urine specimen is evaluated within 1 hour of voiding, otherwise should be kept at 4°C.

Annual screening UAs are not recommended by the American Academy of Pediatrics (AAP) unless patient is at high risk of chronic kidney disease.

URINALYSIS

Urinalysis, usually consisting of the evaluation of physicochemical properties of the urine and sediment examination, has the potential to provide a specific evaluation of the urogenital tract, as well as information concerning systemic changes. While urinalysis is often included in non-clinical toxicology study protocols, sediment examination is more common in diagnostic cases. However, urinalysis is not used optimally in most toxicology studies, because of technical difficulties surrounding sample collection in laboratory species. Thus, urine samples are frequently taken at a single time point, such as at necropsy, rather than collected over a timed period, and determinations are made without reference to urine volume. Sample contamination may also be an issue, due to collection techniques.

In experimental toxicologic pathology, urinary electrolyte excretion determinations, particularly evident in Japanese toxicity studies, and urinary enzyme activities for the identification of renal tubular toxicity, are incorporated in some toxicity study protocols or in special investigations of potential nephrotoxins. If there are no treatment-related microscopic findings in a representative number of high dose animals, there is generally no justification for expending effort in evaluating microscopic sediment from animals in middle- and low-dose groups in toxicity studies. Over-interpretation of results of urine electrolyte excretion, including fractional clearance, should be guarded against in view of the variability among animals, and the relative lack of experience of most laboratories in performing and interpreting these investigations. Numerous studies have evaluated urinary enzymes, including β-microglobulin, ALP, GGT, LDH, lysozyme, and acid phosphatase, as specific indicators of segmental nephron injury. In addition, protein levels and protein:creatinine ratios can be examined.

Variation between laboratories makes comparison of results impossible, due to lack of standardization of methodologies. Urinary enzyme quantification may be useful in investigations designed to examine specific questions regarding nephrotoxic; however, there is no conclusive evidence to indicate that a particular enzyme or enzyme profile is superior over others as a screening tool for nephrotoxicity in general. The scientific value of conducting urinalysis, in terms of knowledge gained for labor invested, is frequently the subject of question. It is recommended that conduct of urinalysis in toxicity studies be performed under the same guiding principles as for other clinical laboratory parameters. That is, in general toxicity studies where the potential toxicity of the test article is unknown, the urinalysis component of the protocol should be efficiently designed to establish a minimum database of results which can be used as a platform for designing appropriate urinalyses in subsequent studies as required. In initial repeat dose, subacute studies, urinalysis can be used effectively to screen for overt changes such as hematuria, pyuria, glucosuria, bilirubinuria, presence of casts, or abnormal crystalluria, as well as providing general information regarding hydration and concentrating ability via specific gravity or osmolality, acid base balance via pH, and energy balance via ketone evaluation of the animal at that point in time. In subsequent studies, such as a subchronic or special investigative study, more targeted urine determinations, such as metabolite identification and quantification, characterization of abnormal crystalluria, electrolyte fractional excretion or enzymuria determination may be added to the protocol as indicated.

Urinalysis

Urinalysis is an effective method to detect and monitor disease activity in lupus nephritis. Precautions to be considered to ensure the quality of the urine examination include expeditious examination of a fresh, early morning, nonrefrigerated specimen and flagging of specimens from patients at risk of nephritis to improve the chances for a vigilant examination. Hematuria (usually microscopic, rarely macroscopic) with dysmorphic (fragmented or misshapen) cells (Fig. 136.1) indicates inflammatory glomerular or tubulointerstitial disease. Granular and fatty casts reflect proteinuric disease, whereas erythrocyte, leukocyte, and mixed cellular casts reflect nephritic disease. Broad and waxy casts are found in chronic renal failure. In severe proliferative nephritis, urine sediment may contain the full range of cells and casts (“telescopic urine sediment”). Urine sediment abnormalities in active lupus nephritis are typically accompanied by significant proteinuria (more than 0.5 g of protein per 24 hours), although isolated hematuria and/or pyuria appearing in the context of generalized lupus activity, and not explained otherwise, might be an early sign of kidney involvement and dictates diligent follow-up.2

Urinalysis

Urinalysis determinants that support presence of a UTI are: (1) pyuria – usually defined as >5 white blood cells/high power field (WBC/hpf) in a centrifuged specimen, (2) any bacteria per hpf in the unstained uncentrifuged urinary sediment, (3) positive urinary leukocyte esterase test, and (4) positive urinary nitrite test. Finding red and white cell casts in the urinary sediment is unreliable. Sensitivity of routine urinalysis for UTI is calculated to be 82% in children <2 years of age.52,53 Urinary leukocyte esterase is produced by the breakdown of white cells in the urine. Dietary nitrates present in the urine, which are reduced by many gram-negative bacteria, are measured by the urinary nitrite test. Gram-positive bacteria and many non-Enterobacteriaceae and non-enteric gram-negative bacteria do not reduce nitrate. Children who lack renal concentrating ability or who receive large amounts of fluids intravenously and infants (who void frequently) may not have bladder dwell-time to break down WBCs or reduce nitrate. Among urinalysis tests, nitrite is the least sensitive (53%) and leukocyte esterase is the least specific (72%).54

In a general pediatric population, when urinary specimens are properly collected and promptly processed, the combined use of testing for leukocyte esterase, nitrite, and microscopic bacteria has almost 100% sensitivity for detection of UTI by positivity of one or more tests (with mean specificity of one or more positive tests of 70%, range 60% to 92%).54 When all (or leukocyte esterase and nitrite tests) are negative, the negative predictive value approaches 100%, and may be sufficient to avoid culture in many situations.51,55

Urinalysis

Urinalysis is a simple yet efficient tool to diagnose renal disease. Its role in the evaluation of proteinuria and hematuria has been discussed in the previous section. A midstream clean-catch technique is adequate in men.23 When performing urine microscopy, proper care should be exercised in preparing the sample. A fresh sample of 10–15 mL of urine should be centrifuged at 1500–3000 rpm for 5 minutes. The supernatant is then decanted and the sediment agitated in the remaining supernatant. A single drop is applied to a clean glass slide, and a cover slip is applied.23,38

3.6 Issues with Urine Specimen Collection

Urinalysis remains one of the key diagnostic tests in the modern clinical laboratory, and, as such, proper timing and collection techniques are important. Urine is essentially an ultrafiltrate of blood. Examination of urine may take several forms: microscopic, chemical (including immunochemical), and electrophoresis. Three different timings of collection are commonly encountered. The most common is the random or “spot” urine collection. However, if it would not unduly delay diagnosis, the first voided urine in the morning is generally the best sample. This is because the first voided urine is generally the most concentrated and contains the highest concentration of sediment. The third timing of collection is the 12- or 24-hour collection. This is the preferred technique for quantitative measurements, such as for creatinine, electrolytes, steroids, and total protein. The usefulness of these collections is limited, however, by poor patient compliance.

For most urine testing, a clean catch specimen is optimal, with a goal of collecting a “midstream” sample for testing. In situations where the patient cannot provide a clean catch specimen, catheterization is another option, but must be performed only by trained personnel. Urine collection from infants and young children prior to toilet training can be facilitated through the use of disposable plastic bags with adhesive surrounding the opening.

For point of care urinalysis (e.g. urine dipstick and pregnancy testing) any clean and dry container is acceptable. Disposable sterile plastic cups and even clean waxed paper cups are often employed. If the sample is to be sent for culture, the specimen should be collected in a sterile container. For routine urinalysis and culture, the containers should not contain preservative. For specific analyses, some preservatives are acceptable. The exception to this is for timed collections where hydrochloric acid, boric acid, or glacial acetic acid is used as a preservative.

Storage of urine specimens at room temperature is generally acceptable for up to two hours. After this time the degradation of cellular and some chemical elements becomes a concern. Likewise bacterial overgrowth of both pathologic as well as contaminating bacteria may occur with prolonged storage at room temperature. Therefore, if more than two hours will elapse between collection and testing of the urine specimen, it must be refrigerated. Refrigerated storage for up to 12 hours is acceptable for urine samples destined for bacterial culture. Again, proper patient identification and specimen labeling is important to avoid errors in reported results.