LABORATORY PROCEDURES TO DETECT THE PRESENCE OF KIDNEY DISEASE

Urinalysis is an important tool for detecting and differentiating various aspects of kidney disease, which often goes unnoticed as the result of its asymptomatic presentation. Urinalysis can be used to detect and monitor the progression of diseases such as diabetes mellitus, glomerulonephritis, and chronic urinary tract infections.16–18 A typical urinalysis provides information about physical and chemical composition, most of which can be completed quickly and inexpensively by visual observation (volume and color) and dipstick testing. Microscopic urinalysis requires use of a light microscope to determine cellular content as described below. CHEMICAL ANALYSIS OF URINE pH The normal urine pH typically ranges from 4.5 to 7.8, and an elevation above this may suggest the presence of urea-splitting bacteria. In patients with renal tubular acidosis, urine pH is usually >5.5 because of impaired hydrogen ion secretion in the distal tubule or collecting duct. Glucose Glucose is usually not present in the urine because the kidney normally completely reabsorbs all the glucose filtered at the glomerulus. When a patient’s blood glucose concentration exceeds the maximum threshold for glucose reabsorption in the kidney (~180 mg/dL), glucosuria will be present. Routine assessment of glucosuria has been replaced by newer methods of direct blood glucose measurements. Urine glucose testing is now predominantly used as a screening tool for the detection of diabetes. Ketones Acetoacetate and acetone are not normally found in the urine; they are however excreted in patients with diabetic ketoacidosis. They are also present under conditions of fasting or starvation. Typically, values of acetone excretion are reported as small (<20 mg/dL), moderate (30 to 40 mg/dL), and large (>80 mg/dL). Nitrite Nitrite is not usually present in urine. The presence of nitrite is most commonly the result of conversion from urinary nitrate by bacteria present in the urine. The presence of nitrite thus suggests that the patient has a urinary tract infection, commonly caused by gramnegative rods such as Escherichia coli. Although false-positive results are very rare, false-negative results are more common and may be caused by lack of dietary nitrate, reduced urine nitrate concentration as a consequence of diuresis, or infections caused by bacteria such as enterococci and Acinetobacter, which do not reduce nitrate, and pseudomonads, which convert nitrate to nitrogen gas.

immunoglobulin A. Falsenegative tests can be produced by the presence of high levels of protein or ascorbic acid. enzymes. Under normal conditions.5). Dipstick tests are now the most common means to determine in a semiquantitative fashion a patient’s daily urine protein or albumin excretion. sulfonamides. Heme The heme test indicates the presence of hemoglobin or myoglobin in the urine.Leukocyte Esterase Leukocyte esterase is released from lysed granulocytes in the urine. the sulfosalicylic acid test was used as a crude measure of proteinuria. Some of these proteins. False-negative results occur with dilute urine (specific gravity <1. such as the Chemstrip Micral-Test II (Roche Diagnostics). 1+ (30 mg/ dL). contamination of the sample with vaginal secretions (such as blood or heavy mucus discharge). plasma proteins remain in the glomerular capillaries as blood perfuses the kidney and thus do not cross the glomerular basement membrane or enter the urinary space. Smaller proteins (<20 kDa) pass across the glomerular basement membrane but are readily reabsorbed in the proximal tubule. The remainder of the protein in the urine is secreted by the tubules (Tamm-Horsfall. the presence of turbidity indicates the qualitative presence of proteinuria. 2+ (100 mg/dL). in those with highly concentrated urine. False-positive tests can result from delayed processing of the urine sample. This test is performed by adding sulfosalicylic acid to urine and then visually comparing this admixture with a tube of untreated urine.17 Evaluation of urinary protein or albumin is now a standard tool used to characterize the severity of CKD and to monitor the rate of disease progression or regression. in the presence of drugs such as penicillin. apoproteins. or by Trichomonas infection (such as trichomoniasis). Historically. and urokinase) or comprised of smaller proteins such as β2-microglobulin. A positive test without the presence of red blood cells suggests either red cell hemolysis or rhabdomyolysis. such as albumin and globulins are not filtered by the glomerulus as a result of charge and size selectivity (>40 kDa). pus. semen.015) and when proteinuria is caused by nonalbumin or low-molecular-weight proteins such as heavy or light chains or Bence Jones proteins.000 mg/dL). Microalbuminuria test strips. The results of these dipstick tests are graded as negative (<10 mg/dL). is now considered the principal marker of kidney damage. when the dipstick is immersed too long. trace (10 to 20 mg/dL). observation of its presence on at least three occasions over a period of 3 to 6 months.16. and peptide hormones. 3+ (300 mg/dL). Because most dipstick methods are not specific for albumin. its presence is suggestive of urinary tract infection. False-positive results can occur in the presence of alkaline urine (pH more than 7. as well as blood. test strips that are specific for low levels of albuminuria (30 to 300 mg/day) should be employed. or 4+ (>1. or tolbutamide. that is. or vaginal secretions. Protein or Albumin Persistent proteinuria or albuminuria. Increased excretion of these low-molecular-weight proteins in the urine is considered a sensitive marker of tubulointerstitial disease. Portable desktop analyzers such as the Chemstrip 101 Urine Analyzer (Roche Diagnostics) and Clinitek 50 Urine Chemistry Analyzer (Bayer Corporation) can also be used as an alternative to visual urinalysis test-strip evaluation. . Most healthy individuals excrete between 30 and 150 mg/day of total protein consisting of approximately 30 mg/ day of albumin.

excretion of >500 mg per day or an albumin-tocreatinine ratio >500 mg/g. however. An important consideration in the assessment of hematuria is whether the cells are of renal origin. this method requires a high degree of patient compliance and is being replaced by a similarly accurate but less cumbersome technique: calculation of the ratio of protein or albumin (in milligrams) to creatinine (in grams) obtained from an untimed (spot) urine specimen. therefore. Specific Gravity Specific gravity is a measure of urine weight relative to water (1.00) that is performed using a refractometer. The normal ratio is <30 mg albumin or <200 mg protein per gram of creatinine in the urine. secreted by the renal tubules. and the presence of dysmorphic cells suggest renal parenchymal origin either because of damage as they pass through the glomerulus or during exposure to the varying osmotic environments of the tubular lumen. with values between 30 and 300 mg of albumin per gram of creatinine considered to be in the microalbuminuria range. Thus. Normal values range from 1. In the presence of red or white blood cells. Casts are cylindrical forms composed of protein. the protein-to-creatinine ratio can be used. Generally the two values correlate. specific gravity is dependent on water intake and urineconcentrating ability. with or without cells that take the shape of the collecting tubules. where they are formed. is a more accurate measure of the kidney’s ability to make a concentrated urine. Osmolality.16 Positive test results should be repeated. sample collection for casts should occur with the first morning void when the urine is most acidic.030. a 24-hour urine collection with measurement of albumin excretion can be used to further define the degree of albuminuria. such as glucose. casts may dissolve and elude detection.when dipped into a urine sample assume a color ranging from white (0 mg/L) to red (100 mg/L). indicating that the cells were of renal origin. depending on the amount of albumin present in the sample. and crystals. when large quantities of heavier molecules. which is a measure of the number of solute particles in the urine. particularly in patients without an underlying cause for renal disease. Contamination of the sample should also be considered when there are many cells and may be a result of the presence of menses or of inadequate sample collection. Casts without cells are labeled hyaline casts and consist of the Tamm-Horsfall mucoprotein. These tests are used in the assessment of urine-concentrating ability and are most informative when interpreted along with the hydration status of the patient and plasma osmolality. casts may be formed that include the cells. whereas for patients with clinical proteinuria. the specific gravity may be elevated relative to the osmolality. White blood cells may be present in the urine in association with infection or inflammatory conditions. such as interstitial nephritis. Otherwise. However. More than 2 cells per highpower field is abnormal. Solubility of the Tamm-Horsfall protein is increased as urine pH rises. such as diabetes or hypertension. . In patients with a positive protein or albumin dipstick test. Monitoring of the degree of glomerular injury in CKD patients should use the albumin-tocreatinine ratio. Microscopic Analysis of Urine Formed elements that may be detected in the urine include erythrocytes and leukocytes. are in the urine.003 to 1. that is. They are nonspecific and may appear in concentrated urine. More than 1 cell per high-power field is usually considered abnormal. casts.

Creatinine is not reabsorbed to any significant extent by the kidneys. 3. a colorimetric method based on the reaction of creatinine with alkaline picrate. For all groups. or an increase of the blood urea nitrogen to a greater extent than the serum creatinine. therefore. This nonspecific method also reacts with noncreatinine chromogens in the serum. Creatinine is a product of creatine metabolism from muscle. 44–2). Serum Creatinine Creatinine is the standard laboratory marker for the detection of kidney disease. The report also noted that among community-dwelling adults. which permits them to be identified with microscopy. Creatinine is eliminated primarily by glomerular filtration. The third National Health and Nutrition Examination Survey (NHANES III) revealed a mean serum creatinine of 0. Many of these have a unique crystalline form.96 mg/ dL in women. calcium magnesium ammonium pyrophosphate. This condition is evident when a patient exhibits prerenal azotemia.0 mg/dL. most of which use the nonspecific Jaffe reaction. Serum or Blood Urea Nitrogen Amino acids metabolized to ammonia are subsequently converted in the liver to urea. At steady state. There is presently no accepted single standard for an “abnormal” serum creatinine.16 mg/dL in men in the United States. and higher among non. Although the serum creatinine concentration alone is not an optimal measure of kidney function. and age-dependent.5 mg/dL for males and females. The reabsorption rate of urea is predominantly dependent on the reabsorption of water. the production of which is dependent on protein availability (diet) and hepatic function. The excretion of urea may.19 Values were lower among Mexican Americans.8 million have a serum creatinine greater than 2. as it is gender.5 to 1. Urea undergoes glomerular filtration followed by reabsorption of up to 50% of the filtered load in the proximal tubule. and an elevated ratio is suggestive of a decreased effective circulating volume. and 0.0 million have a serum creatinine greater than 1. urea reabsorption. calcium phosphate. The normal blood urea nitrogen-to-creatinine ratio is 10 to 15:1. and cystine. calcium oxalate. which stimulates increased water.9 million have a serum creatinine greater than 1.A variety of crystals may be present in the urine. The concentration of creatinine in serum is a function of creatinine production and renal excretion. the serum creatinine concentration rises (Fig. 10. be decreased under conditions which necessitate water conservation such as dehydration although the GFR may be normal or only slightly reduced. Several methods are used for the determination of the serum creatinine concentration. Despite these limitations. which may . it is often used as a marker for referral to a nephrologist. the blood urea nitrogen is usually used in combination with the serum creatinine concentration as a simple screening test for the detection of renal dysfunction. its production is directly dependent on muscle mass. race. and hence. and 1.5 mg/dL. and as GFR declines. therefore.7 mg/dL. the “normal” serum creatinine concentration range is 0. including uric acid.Hispanic blacks. the serum creatinine increased with age.

cephalexin. However. Diabetic ketoacidosis may produce increased concentrations of acetoacetate. because the concentration of creatinine in urine is several-fold greater than the serum concentration. cefoxitin.17 The noncreatinine chromogens are not present in the urine in sufficient quantities to interfere with the urinary creatinine measurement. some cephalosporin antibiotics are associated with a false increase in the serum creatinine concentration. and . CLcr has been proposed to serve as a good measure of GFR in subjects with normal renal function. Scr = serum creatinine concentration. This “normal” interference results in an increase in the serum creatinine concentration of approximately 10% and thereby the measured creatinine clearance would underestimate the true GFR by 10%. including cephalothin. which serves as a chromophore in the Jaffe reaction. In subjects with normal renal function. The impact of this interference is seen with the creatinine clearance (CLcr) calculation: CLcr = (Ucr × V) / Scr × t) where Ucr = urine creatinine concentration.20 This becomes of major importance when kidney function is reduced to less than 50% of normal. due to the increasing contribution of tubular secretion to the renal clearance of creatinine. fructose. which increases urine creatinine by nearly 10%. protein. In addition. this false increase in serum creatinine becomes less noticeable as the true creatinine concentration rises. this tends to counterbalance the effect of the contribution of tubular secretion of creatinine. cefaclor. pyruvate. Other substances that also react with this procedure in the serum include glucose. V = urine volume. Thus. and t = duration of the urine collection. and ascorbic acid (Table 44–3). thereby increasing the serum creatinine concentration. cefazolin.result in a falsely increased serum creatinine concentration. uric acid.

The interference is concentration dependent and results in a 10% to 100% decrease of the serum creatinine concentration. ofloxacin.cephradine. as the result of minimal muscle mass. These differences emphasize the need to standardize a method within the clinic setting and require the clinician to be aware of the methods employed in the laboratory for the determination of creatinine concentrations. creatinine excretion rate.27 Ingestion of creatine as an ergogenic dietary supplement is currently popular. Serum creatinine concentrations may rise as much as 50% within 2 hours of a meat meal and remain elevated for as long as 8 to 24 hours.22 The degree of interference is dependent on the serum concentration of the antibiotic.29 however. The problem is most evident when blood samples are contaminated with residual IV solution containing the interfering drug. levofloxacin.23 Daly et al. given as a single 400-mg dose can result in a reduction of the CLcr-to-inulin clearance ratio from 1. Robinson et al. Poortsmans et al. The authors hypothesized that both drugs compete with the chromogenic dye for oxidation by hydrogen peroxide in a concentration-dependent manner. Other factors that influence the serum creatinine concentration include the dietary intake of creatine. These interferences are not observed when the serum creatinine is measured using an enzymatic technique. and temafloxacin). Cimetidine. but does not interact with the Jaffe method. does not have a similar effect on creatinine secretion following single doses of 300 to 1. Ranitidine. an H2-receptor antagonist similar to cimetidine. do not produce a false elevation in serum creatinine. Its formation rate depends on the zero-order production from creatine metabolism. individuals with more muscle mass have a higher serum creatinine concentration at any given degree of kidney function than those with less muscle mass. so blood samples for creatinine should be obtained when the antibiotic concentration is lowest (at the end of a dosing interval). Strenuous exercise is associated with an increase of approximately 10% in the serum creatinine concentration. as do those with spinal cord injuries. or creatinine clearance. fleroxacin. During the cooking of meat. as well as input from other sources. which compete for creatinine secretion at the cationic transport system in a dosedependent fashion.200 mg. without a change in inulin clearance.24 reported a false-negative effect of dobutamine and dopamine on the serum creatinine value when measured using the Ektachem system. or formation rate. will have very low serum creatinine concentrations. Other compounds are known to interfere with the serum creatinine concentration by inhibition of the active tubular secretion of creatinine. such as dietary intake. In contrast.28 evaluated a short-term regimen of 20 g creatine per day for 5 days in healthy subjects. The antifungal agent 5-flucytosine causes an increase in the serum creatinine when measured using the Ektachem enzymatic system. Creatine metabolism is directly proportional to muscle mass. therefore.26 Elderly patients and those with poor nutrition may also have low serum creatinine concentrations (<1. some creatine is converted to creatinine. reported a 25% to 40% increase in the serum creatinine . such as the fluoroquinolones (ciprofloxacin. There are conflicting reports as to the effect of creatine ingestion on the serum creatinine concentration.21 whereas other antibiotics. however. Among these are cimetidine and trimethoprim. and “output” function. sparfloxacin.. and reported no significant change in the serum creatinine.30 to 1. lomefloxacin.03.25 The serum creatinine concentration is dependent on the “input” function. cachectic patients.0 mg/dL) secondary to decreased muscle mass. which is rapidly absorbed following ingestion. or elimination rate.

It is freely filtered at the glomerulus and undergoes some reabsorption and catabolism in the proximal tubule. The issue of whether creatine inges. it has been recommended as a test of kidney function because of findings that serum concentrations significantly correlated with GFR as well as serum creatinine. In a 4year longitudinal study in 30 Pima Indians without CKD. comparison of 125I-iothalamate with serum creatinine and cystatin C showed that the serum creatinine began to increase when the GFR was 75 mL/min/1. Although the fluctuation is minimal. These conditions present a problem only when a single serum creatinine concentration is used to represent the entire 24-hour collection period. This is rarely done in clinical practice. it has been shown that serum cystatin C concentrations can be altered by many factors other than kidney function. Early evidence suggests that it may be a more sensitive index of kidney disease. as long as the patient has stable kidney function. and interconversion between creatinine and creatine that can occur if the urine is not maintained at a pH <6.31–33 Originally introduced in Europe. the reciprocal cystatin C index (100/cystatin C) was shown to be a better predictor of declining GFR than was reciprocal .38 mg/L in children older than 1 year of age. To minimize this effect.31 However. which may limit its use in longitudinal evaluations of renal function. serum C-reactive protein levels. and 0. Collection of urine remains a limiting factor in the 24-hour CLcr because of incomplete collections. which is usually the case. An alternative is to obtain multiple samples and calculate the area under the serum concentration time curve and divide this by the collection time interval to obtain the average plasma creatinine concentration. suggesting that creatine supplementation may be a risk factor in patients with preexisting renal disease. and rheumatoid arthritis. the CLcr is usually performed over a 24-hour period with the plasma creatinine obtained in the morning. steroid therapy. nutritional status. Keevil et al.21 mg/L in adults. However.34 reported that serum cystatin C had greater intraindividual variability than serum creatinine in healthy individuals. height.30 They noted that creatine supplementation led to an increase in renal disease progression in a rat model for renal cystic disease. Cystatin C Cystatin C is a 132-amino-acid (13.54 to 1.73 m2 as compared to 88 mL/min/1. Its sensitivity as an indicator of renal function relative to other markers such as serum creatinine is yet to be fully defined. The recent development of an automated immunoassay technique has resulted in the development of suggested reference ranges of 0. Several recent studies suggest that there is still much to discover before we know whether this test will be a replacement for creatinine or a confounding ancillary test.73 m2 for cystatin C.tion adversely affects kidney function was studied by Edmonds et al. Diurnal variation in serum creatinine concentration may also affect the accuracy of the CLcr determination. The renal excretion rate of creatinine was not measured. but points out the need to question patients regarding dietary intake for the 24 hours preceding the measurement of CLcr. cigarette smoking. such as age. gender. weight. whereas the nadir is in the morning.concentration after ingestion of 20 g creatine per day for either 5 days or 8 weeks. the observed peak plasma creatinine concentration generally occurs at approximately 7:00 PM.3-kDa) cysteine protease inhibitor produced by all nucleated cells of the body that is being investigated as a potential biomarker of renal function.70 to 1.

in pediatric and adult renal transplant recipients. possibly due to the formation of cystatin–immunoglobulin complexes or reduced tubular catabolism.36 reported increased cystatin C concentrations that were independent of CLcr. Cystatin C concentrations were also noted to be independent of GFR and weakly associated with measured creatinine clearance.37. . or CLcr. estimated GFR.35 However in patients being treated for malignant disease.40 It appears that cystatin C is a nonspecific marker of GFR that likely is a more sensitive index than serum creatinine of early changes in GFR. suggesting that it may provide useful prognostic information in some populations. 39.serum creatinine. Page et al.38 Two additional studies have shown a strong association between serum cystatin C and cardiovascular disease in elderly and non-CKD patients. Further longitudinal evaluations will be necessary to clarify its usefulness as a prognostic index of trends in renal function and/or cardiovascular outcomes.