Urinalysis Chemical Composition
Bilirubin & Urobilinogens
Basic Science Bilirubin is a byproduct of red blood cell (RBC) breakdown in the reticuloendothelial system. Hemoglobin is converted to unconjugated bilirubin, which is then released into the blood stream. There it is reversibly bound to albumin and carried to the liver, where hepatocytes remove it by carrier-mediated active transport. Hepatocytes conjugate bilirubin with glucuronic acid to produce a water-soluble compound. The liver excretes this as conjugated bilirubin, a constituent of bile. It is passed into the small intestine where it is converted back to its unconjugated form and reduced by the gut flora to urobilinogen. Most of this urobilinogen is reduced to stercobilinogen and in the large intestine both compounds are oxidized to urobilins and stercobilins. Approximately 50% of urobilinogen is reabsorbed into the enterohepatic circulation and taken to the liver, which re-excretes it. A small amount (~1–4 mg/day), however, remains in the bloodstream and is excreted by the kidneys in urine.
Clinical Considerations Normal urine contains only small amounts of urobilinogen. Elevated levels may indicate hemolysis and hepatocellular disease associated with pregnancy-related conditions such as hemolysis, elevated liver enzymes and low platelet count (HELLP).[15,16] Bile duct obstruction can decrease levels of urobilinogen. False-positives may be due to elevated nitrite levels or phenazopyridine, a drug commonly used as a urinary dye for diagnostic purposes and to decrease bladder irritation associated with certain conditions such as a UTI.
Only conjugated bilirubin (water soluble) may be passed into urine in pathologic states. Reagent test strips are very sensitive to bilirubin, detecting as little as 0.85 µmol/l. Presence of bilirubin in urine indicates either bile duct obstruction (e.g., common bile duct stones or cholestasis of pregnancy) or intrinsic hepatic disease. The presence of bilirubin in urine may precede clinically apparent jaundice. However, false-positive test results can occur if urine is contaminated with stool and by phenazopyridine. Prolonged storage, and exposure to light, selenium and chlorpromazine can lead to false-negative results.
In summary, excess levels of urobilinogen and bilirubin in the urine may signal liver disease, and more research is needed to correlate these levels with the onset and severity of clinical disease. In addition, with the introduction of liver enzyme measurements, the clinical utility of detecting these substances in urine has decreased.
Basic Science Detection of glycosuria by dipstick is based on a double-oxidative reaction that is specific to glucose, and there is no crossreactivity with other sugars. In the first step, urine glucose reacts with glucose oxidase on the dipstick to form gluconic acid and hydrogen peroxide. The hydrogen peroxide then causes oxidation of the chromogen on the dipstick and a color change is produced.[2,20]
Glucose is normally filtered by the glomerulus, but is almost completely reabsorbed in the proximal tubule. Most reagent strips use a glucose oxidase/peroxidase method, which generally detects levels of glucose as low as 2.8 mmol/l. The renal threshold for glucose is generally 8.9–9.9 mmol/l, therefore the presence of detectable urine glucose indicates blood glucose in excess of 11.6 mmol/l.[2,15,20] Large quantities of ketones, ascorbate and phenazopyridine metabolites may interfere with the color reaction. Urine peroxide contamination, levodopa metabolites, diuretics and birth control pills may cause false-positive results. As a screening test for diabetes, fasting urine glucose testing has a specificity of 98% and a sensitivity of 17%.
Clinical Considerations Dipstick measurement of glucose was once used to monitor diabetic therapy. However, blood glucose measurement by finger-stick method is a more accurate monitor than urine glucose. Urine glucose is dependent on urine volume, and blood glucose elevations usually precede the appearance of glucose in the urine.
Trace glycosuria can be a normal finding in pregnancy. Routine urine screening for glucose at each prenatal visit is the traditional standard of care. Gribble et al. examined this in a retrospective review of 2965 women, and found that patients with glycosuria in the first and second trimester had a significantly higher incidence of developing gestational diabetes than those whose urine tested negative for glucose (12.8 vs 2.9%; p = 0.003). However, for women without evidence of gestational diabetes, no other clinically important differences such as hypertension, placental abruption or premature delivery could be measured. This suggests that once the more definitive glucose tolerance test is performed at 24–28 weeks of gestation, those who test negative may not benefit from ongoing urine glucose screening in the third trimester.
In summary, the value of reagent strip glucose is now limited almost entirely to screening for hypergylcemia in pregnancy, and consistently abnormal results should prompt a formal evaluation of glucose tolerance.
Basic Science Ketones are products of body fat metabolism and are not normally found in urine. The three physiologic ketones are acetoacetic acid, acetone and β-hydroxy-butyric acid. The reagent strip test uses a nitroprusside reaction, in which sodium nitroferricyanide and glycine react with acetoacetatic acid and acetone at alkaline pH to yield a violet-colored product. Interestingly, β-hydroxybutyrate (often 80% of total serum ketones present in ketosis) is not normally detected by the nitroprusside reaction. Acidic urine, elevated USG, and phenazopyridine and levodopa ingestion can give false-positive results. False-negative results can occur if there is a delay in examination of the urine specimen.
Clinical Considerations The presence of ketones in urine is indicative of a gluconeogenic metabolic state that can occur with pregnancy, starvation, low-carbohydrate diets, febrile illness or uncontrolled diabetes. In labor, ketonuria is a common occurrence due to physiologic stress compounded by decreased oral intake. For many years, it has been debated whether ketosis and ketonuria are normal physiologic processes in labor, or whether it requires intervention. A higher incidence of ketosis has been described for nulliparous women.[24,25] Adverse effects may include increased likelihoods of augmentation of labor, forceps-assisted delivery and postpartum blood loss.[24,25] Although ketones do cross the placenta, effects on the fetus are unknown.
A 2008 Cochrane Review attempted to assess the effects of intravenous fluids or increased oral intake on maternal, fetal and neonatal outcomes in labor for the treatment of ketosis compared with no intervention. Unfortunately, the authors were unable to find any studies that met their inclusion criteria, and hence no recommendations could be made.
Ketonuria is also a hallmark of hyperemesis gravidarum, along with frequent vomiting periods, dehydration with electrolyte disturbances and loss of >5% prepregnant weight. It can be useful as a guide to therapy, as it is expected to resolve with fluid and caloric replacement.
In summary, ketonuria is associated with starvation or stress states, medications and certain disease states such as infection, diabetes and hyperemesis gravidarum.
Basic Science The American Urological Association defines microscopic hematuria as three or more RBCs per high power field (HPF) on microscopic evaluation of urinary sediment from 2–3 different urinary specimens.[28,29] The fastest and simplest way to detect microscopic hematuria is with the urine dipstick. Detection of hemoglobin with the urine dipstick depends on the fact that hemoglobin catalyzes the reaction between hydrogen peroxide and a chromagen, which are impregnated on a cellulose strip. The resulting reaction causes the chromagen to turn green, with the degree of color change directly related to the amount of hemoglobin present in the urine.
A urine test strip for blood can detect as little as 150 µl/l of free hemoglobin, corresponding to 5–20 RBC/l on microscopic analysis, or 1–4 RBC/HPF of concentrated urine sediment. It has been shown to be 91–100% sensitive and 65–99% specific for detecting more than 3 RBC per HPF. False-positive results occur in the presence of myoglobin, free hemoglobin or porphyrins. False-positive results can also occur with dehydration and when oxidizing agents are present (such as povidone-iodine and hypochlorite). False-negative results can occur if the urine has high concentrations of ascorbic acid or if it had prolonged exposure to air before testing. When gross hematuria is evident, the urine should be centrifuged. True hematuria is present if the sediment contains RBCs and the supernatant is clear. A red supernatant that is negative for hemoglobin is called pseudohematuria.
Clinical Considerations The clinical significance of hematuria can range from a minor incidental finding that does not require further work-up to a sign of significant disease such as a renal or urinary malignancy. Physical examination should include speculum examination to rule out a genital source for the bleeding. The type and extent of evaluation should be based on the presence of risk factors and accompanying symptoms. Transient microscopic or macroscopic hematuria can appear after vigorous exercise, but exercise-induced hematuria should be a diagnosis of exclusion and hematuria must resolve within several days.
Risk factors for urologic malignancy include: age >40 years; smoking; previous urologic disease; history of pelvic irradiation; occupational exposure to chemicals or dyes; and analgesic abuse.[28,29] When malignancy is suspected, further investigations with urine cytology, cystoscopy and upper urinary tract imaging are recommended. Intravenous urography (IVU) has traditionally been considered to be the best initial study for the evaluation of microhematuria. However, IVU by itself has limited sensitivity in detecting small renal masses and cannot distinguish solid from cystic masses. Further lesion characterization by ultrasonography, computed tomography or MRI is necessary.[28,29] Not enough data exist to show the impact of IVU, ultrasonography, computed tomography or MRI on the management of patients with microscopic hematuria, and evidence-based imaging guidelines have not been formulated. In 2009, the Canadian Urological Association recommended that after accounting for patient safety, test availability and cost, ultrasonography should be the imaging modality of first choice. However, the overall incidence of urinary tract cancer is low in patients with hematuria (0.68% over 3 years). A large population-based cohort study established that microscopic hematuria had 50% sensitivity, 84% specificity and 1.3% positive predictive value in the detection of urinary tract cancer. In this study, older age (greater than 40 years; odds ratio [OR]: 17.0; 95% CI: 11.2–25.7), greater hematuria (greater than 25 RBC per HPF; OR: 4.0; 95% CI: 3.5–4.5) and male gender (OR: 4.8; 95% CI: 4.2–5.6) were associated with a higher risk of cancer.
Fever, urgency, pain and frequency can indicate infection. Dipstick hematuria has good sensitivity (68–92%) but lesser specificity (up to 46%) for UTI detection. Microscopic hematuria has also been documented in neuritis-associated voiding dysfunction with herpes zoster infection (in the absence of herpes cystitis, which caused pyuria). An inflammatory bladder reaction such as interstitial cystitis/painful bladder syndrome may cause hematuria. Interstitial cystitis/painful bladder syndrome is more common in women and a high level of suspicion may be indicated with relevant symptoms of pain, urgency and frequency.
In pregnancy, hematuria may be caused by a variety of conditions. Infection is the most common, and is followed in decreasing order of incidence by kidney stones, underlying renal diseases, medications, trauma and tumors. UTI in pregnancy is associated with increased complications for both mother and fetus. Any suspicion of UTI in pregnancy warrants a formal urine culture and sensitivity testing and careful documentation of resolution following treatment. The incidence of nephrolithiasis in pregnant women is similar to nonpregnant women, at approximately 1 in every 2000 pregnancies. Microscopic or gross hematuria has been reported in up to 95% of these cases. Acute renal disease can occur de novo in pregnancy and may present solely with hematuria. The usual causes are glomerulonephritis, interstitial nephritis or lupus nephritis. Placenta percreta can be an obstetric cause of hematuria if it invades the bladder.[39,40] In a review of 54 reported cases of placenta percreta invading the urinary bladder, hematuria was the initial presenting complaint in 31% of patients (17 out of 54). Hematuria as a result of renal blunt trauma can be a presenting sign in pregnant women who are victims of domestic violence, which is more common in pregnancy.
In rare cases, hematuria in association with proteinuria may be part of a paraneoplastic syndrome. Vasculitis, along with hematuria and proteinuria, were found in a patient with an ovarian malignancy. After surgical resection, urinalysis findings slowly improved.
In summary, microscopic hematuria may be completely benign or associated with life-threatening conditions, and high-risk patients should undergo careful evaluation for malignant urinary diseases.
Leukocyte Esterase & Nitrites
Basic Science The esterase method of detecting leukocytes in a urine sample relies on the fact that esterases are released from lysed urine granulocytes (neutrophils). In the dipstick method, neutrophilic esterases catalyze the hydrolysis of esters to produce their respective alcohols and acids. The alcohols produced then react with a diazonium salt to produce a pink to purple color. The intensity of the color produced is proportional to the amount of enzyme present, which is related to the number of neutrophils present. If urine is allowed to stand, a greater number of leukocytes will lyse leading to a more intense reaction. High levels of glucose, albumin, ascorbic acid, tetracycline or cephalexin, or large amounts of oxalic acid, may inhibit the reaction and lead to false-negative results. Vaginal contamination can produce false positives. In one study comparing urine dipstick to microscopy performed by a nephrologist in the detection of white blood cells (WBCs) in urine, the sensitivity and specificity for leukocyte esterase was found to be 81.0 and 64.3%, respectively.
Many bacteria that are potential urinary pathogens are able to reduce nitrates to nitrites. Common organisms include Escherichia coli, Klebsiella, Enterobacter, Proteus, Staphylococcus, and Pseudomonas species. However, some bacteria such as Enterococcus spp., Streptococcus faecalis, Neisseria gonorrhea and Mycobacterium tuberculosis cannot produce nitrites. False-negative results are common and may be due to low levels of urine nitrates resulting from low dietary intake. It should also be remembered that approximately 4 h are needed for bacteria to make the conversion, and hence frequent urination may also lead to false-negative results.
Clinical Considerations Several studies looking at various clinical settings and patient populations have evaluated the utility of urinalysis to predict UTIs. D'Souza and D'Souza collected 100 midstream urine samples from pregnant patients with symptoms such as dysuria, urinary frequency, loin pain, nonspecific abdominal pain and threatened preterm labor, suggestive of a UTI. Dipstick urinalysis was compared with laboratory culture. A positive nitrite test on urinalysis was the only parameter that correlated with culture-proven UTI. Mignini et al. compared dipstick urinalysis to urine culture in 3032 pregnant, asymptomatic patients. When either leukocyte esterase or nitrites were positive on urinalysis, the respective sensitivity and specificity to detect a UTI was 53 and 92%. Since organisms that are not uropathogens also produce leukocyte esterase, it has a greater sensitivity (up to 97%) but lesser specificity (41–86%) than nitrites for UTI detection. When positive nitrites were combined with symptoms of a UTI in a urogynecologic population undergoing cystometry, the sensitivity of a nitrite-positive test to detect a UTI increased from 40 to 89% and the negative predictive value was 96%. The authors concluded that symptom-negative, nitrite-negative patients do not need a urine culture, thus increasing the cost–effectiveness of UTI screening in urogynecologic patients.
Urinalysis is also commonly used to rule out a UTI in the general gynecologic population, particularly in those patients presenting with voiding dysfunction and/or pelvic organ prolapse. Raza-Kahn et al. retrospectively reviewed the urine samples of 143 women presenting with urogynecologic complaints. Dipstick urinalysis was considered positive in the presence of either nitrites or leukocyte esterase. When compared with urine culture, the respective sensitivity and specificity of dipstick urinalysis was 35 and 98%. When patients with pelvic organ prolapse were analysed separately (n = 122), the sensitivity fell to 29% while the specificity remained high at 98%. These results demonstrate the importance of obtaining a urine culture despite a negative urinalysis in this population. Unfortunately, many laboratories will not perform urine culture unless the urinalysis is positive.
In summary, the presence of nitrites and/or leukocyte esterase has good specificity for the diagnosis of a UTI in both pregnant and nonpregnant patients, and should prompt further evaluation via urine culture. Empiric treatment seems prudent in selected cases, such as in pregnancy, the pediatric population and in symptomatic patients, where the presence of leukocyte esterase and/or nitrites predicts a positive culture with reasonable accuracy. Optimal antibiotic choice and length of therapy in the treatment of uncomplicated UTI in pregnancy has been difficult to establish. A recent meta-analysis of ten randomized controlled trials failed to demonstrate a significant difference among several types of antibiotics. All of them were shown to be highly effective and have a low incidence of adverse effects.
Basic Science Dipstick analysis provides a semiquantitative measure of the urine protein concentration. Proteins in solution interfere with the dye–buffer combination, causing the yellow panel to turn green. False-positive results occur: with alkaline urine (pH >7.5); when the dipstick is immersed too long; with highly concentrated urine; with gross hematuria; in the presence of penicillin, sulfonamides or tolbutamide; and with pus, semen or vaginal secretions. False-negative results occur with dilute urine (USG more than 1.015) and when the urinary proteins are nonalbumin or have low molecular weight. The results are graded as negative (less than 10 mg/dl), trace (10–20 mg/dl), 1+ (30 mg/dl), 2+ (100 mg/dl), 3+ (300 mg/dl) or 4+ (1000 mg/dl). This method preferentially detects albumin and is less sensitive to globulins or parts of globulins (heavy or light chains or Bence–Jones proteins).
Clinical Considerations Community-based cohort studies show that proteinuria in dipstick urinalysis predicts long-term risk of chronic and end-stage kidney disease, and cardiovascular, cancer-related and all-cause mortality.[52–55] When trace and 1+ were considered together as mild proteinuria in a study investigating mortality risk in a Canadian cohort of nearly 1 million individuals, the all-cause mortality risk was 2.1. Albuminuria was associated with a 8.3-fold likelihood of a diagnosis of bladder cancer and a 2.4-fold likelihood of a diagnosis of lung cancer in a large, low-risk, population-based longitudinal study of over 5000 participants. Trace proteinuria affects 6–9% of the adult population and is associated with a lifespan shortening of up to 7 years. Two-thirds of patients with chronic kidney disease have proteinuria. However, fewer than 2% of patients whose urine dipstick test is positive for protein have serious and treatable urinary tract disorders. Currently, clinical practice guidelines for proteinuria screening limit testing to the elderly and to high-risk patients (with diabetes and hypertension), and differ in their recommendations regarding the utility of dipsticks for identifying proteinuria in asymptomatic individuals.[54,57,58]
Proteinuria has additional significance during pregnancy. Dipstick protein measurement is the recommended screening tool for proteinuria in pregnancy.[59,60] Urinary protein excretion is considered abnormal in pregnant women when it exceeds 300 mg/24 h at anytime during gestation, a level that usually correlates with 1+ on a urine dipstick. Proteinuria documented before pregnancy or before 20 weeks of gestation suggests a pre-existing renal disease, while preeclampsia is the leading diagnosis that must be excluded in all women with proteinuria first identified after 20 weeks of gestation. The sensitivity of a result ≥1+ protein on a dipstick measurement for predicting proteinuria of 300 mg/24 h varies in different studies, ranging from 47 to 86%, with specificity ranging from 39 to 95%. In a systematic review that summarized results from seven prospective studies, Waugh et al. concluded that the accuracy of dipstick urinalysis with a 1+ threshold in the prediction of significant proteinuria is poor. The Society of Obstetricians and Gynecologists of Canada defines heavy proteinuria in pregnancy as >3–5 g/day, and this serves as a marker for the diagnosis of severe preeclampsia. However, studies in recent years have shown that isolated proteinuria does not correlate with maternal or fetal complications, and that the amount of proteinuria does not predict severity of maternal or fetal outcomes in women with preeclampsia. Nevertheless, dipstick testing is still the fastest detection method and a positive dipstick for protein in a pregnant woman should not be disregarded. In addition, a negative or trace value should not be ignored in a patient with new hypertension. Up to 12% of negative/trace results are false-negatives when followed by a 24-h urine collection. Therefore, a practical approach would be to proceed to 24-h urine collection or spot protein to creatinine ratio measurement if dipstick analysis is positive for protein on at least two different occasions or in the setting of new hypertension or other features suggestive of preeclampsia.
The spot protein to creatinine ratio has become widely accepted as an accurate alternative to the 24-h urine collection in quantifying the degree of proteinuria in pregnancy. A cutoff of ≥30 mg/mmol urinary creatinine has been recommended by The Society of Obstetricians and Gynecologists of Canada. Among pregnant women with hypertension, this value has a sensitivity of 85% and specificity of 76% in diagnosing clinically significant proteinuria.
In summary, proteinuria on dipstick urinalysis is used for screening and initial detection of preeclampsia in pregnancy, and may predict morbidity and mortality in other patient populations.
Expert Rev of Obstet Gynecol. 2012;7(3):269-279. © 2012 Expert Reviews Ltd.