Medical Laboratory Observer

Laboratory evaluation of discolored urine

By Rebecca M. Hsu, MD, and Leland B. Baskin, MD

When is it hematuria?

Many factors, both pathologic and nonpathologic, may alter urine color.^1–10 Given the large number of possible causes for change in urine color, it is important to determine if that cause is pathologic. Red urine is a source of concern because this discoloration may represent hematuria, which suggests more serious genitourinary or systemic pathology. When is this a symptom of hematuria? To determine this, you should be familiar with the causes of and laboratory evaluation techniques for hematuria. 

Normal urine is generally clear, with a yellow-amber color caused by urochrome, uroerythrin, or urobilin. Concentration of the urine will determine the color intensity. The presence of myoglobin and hemoglobin and its metabolites (bilirubin, biliverdin, and urobilinogen) will change urine color from the normal yellow-amber to colors that range from pink-red to purple, brown, or black. The darker colors, such as brown or “tea-colored” urine, suggest that the problem lies higher in the urinary tract, such as within the renal glomerulus. Pink or red urine is generally seen in diseases affecting the lower urinary tract. 

Hematuria is a term often used to refer to any red-brown urine specimen. By definition, however, hematuria refers to the presence of blood [greater than 3 intact erythrocytes per high power field (hpf)] in the urine. Generally, in the healthy kidney, blood cells are too large to be filtered through the glomerulus. Although a few cellular components might be present in the urine of healthy individuals, hematuria is usually considered an abnormality that indicates acute or chronic renal or urogenital disease. Two other states that may mimic the red-brown color seen in hematuria are hemoglobinuria and myoglobinuria. Hemoglobinuria and myoglobinuria refer strictly to the presence of hemoglobin and myoglobin, respectively, in solution in urine. True hematuria is one of the most common abnormalities detected on urinalysis, while hemoglobinuria is less common, and myoglobinuria is rare. Treatment for these diseases vary, making the distinction between them crucial for patient care.^1–3,5

History and physical examination are the essential first steps in the diagnosis. Hematuria may be found in several types of familial and congenital diseases, such as benign familial hematuria, polycystic renal disease, Alport’s syndrome (hereditary progressive nephritis), collagen vascular disease (systemic lupus erythematosus), and several metabolic diseases. However, these diseases are relatively rare and can generally be excluded with a family and medical history. 

Lab evaluation of discolored urine

Specimen collection and processing. Changes occur in unpreserved urine at room temperature in approximately 1 hour; therefore, urine should be examined within 1 hour or refrigerated at 48C for optimal results. Among the changes that affect the apparent or real hemoglobin concentrations of urine within 1 hour at 208C are: 

1. an increase in pH from breakdown of urea to ammonia by urease-producing bacteria (change in pH may affect detection of myoglobin),
2. a decrease in bilirubin from light exposure, and 
3. changes in color caused by oxidation or reduction reactions, including oxidation of urobilinogen to urobilin.⁶

Urine examination. Although this discussion focuses on the evaluation and causes of discolored urine, it must also be kept in mind that hematuria can exist without visual color change in the urine, thus requiring other laboratory methods for urine examination. In addition to gross examination, urinalysis and urine microscopy for evaluation of urine sediment may also be required. 

Urine dipstick. Because visual examination of the urine is insufficient for diagnosis, a reagent strip commonly called the “urine dipstick” is used as a primary screening test. The dipstick test for hemoglobin is based on the liberation of oxygen from peroxide in the reagent strip by the peroxidase-like activity of heme from lysed erythrocytes, free hemoglobin, or myoglobin.³ Intact erythrocytes are lysed on the strip, allowing the heme to be released. Well-mixed urine is essential because intact erythrocytes might be missed if only supernatant is used. Heme catalyzes the oxidation of tetramethylbenzidine, which changes the yellow test zone to a blue-green color. The dipstick is highly sensitive in detecting minute amounts of hemoglobin and myoglobin (0.02–0.03 mg/dL, respectively).⁵ Specific gravity of 1.010 or less in urine may cause lysis of erythrocytes, as does alkaline pH. Increased specific gravity and the medication captopril both decrease reactivity and may lead to false-negative test results, while oxidizing agents, such as hypochlorite, ascorbic acid (vitamin C), and microbial peroxidase (from a bacterial infection) may cause false-positive test results.^6,7,10 

Additional tests. Chemical tests may be performed to rule out myoglobin and urobilinogen. A qualitative test for myoglobin may be performed by adding 2.8 g of NH4SO4 to 5 mL urine, which causes 80% saturation and precipitation of hemoglobin that can then be removed by filtration or centrifugation. The supernatant appears normal when hemoglobin is present but remains dark red when myoglobin is present. Table 2 lists the findings that aid in differentiating hematuria from hemoglobinuria and myoglobinuria.^3,11-13

A modified Ehrlich’s reaction is used to detect urobilinogen, in which the urobilinogen is oxidized to urobilin on exposure to air. Urobilinogen should not be present in quantities greater than 1.0 mg/dL. Elevation may indicate biliary obstruction, hepatocellular damage, cirrhosis, or hemolysis. False- positive test results may occur with methyldopa, sulfonamides, azo dyes, and warm urine. 

Urine microscopy. The microscopic constituents present in urine sediment may suggest the location of the abnormality responsible for discolored urine or urine that was not discolored but results of a urine dipstick were positive for hematuria. Because some of these constituents may dissolve with time, a freshly voided specimen is required for microscopic examination.

Generally, 10–15 mL of urine should be centrifuged at 1,500 rpm for 5–7 minutes, supernatant removed, and sediment examined under dry, high-power magnification of 10–403. The average number of red blood cells per hpf is counted, and the presence of greater than 3 RBCs/hpf is accepted as hematuria, although nearly 3% of the normal population will excrete more than 3 RBCs/hpf.¹⁴ A healthy individual excretes approximately 1,000 RBCs/mL of urine, with the upper limits of normal ranging from 5,000–8,000 RBCs/mL.⁸ The significance of these and other cells in the urine are outlined in Table 3. 

The shape of erythrocytes under phase-contrast microscopy also provides a clue to the origin of hematuria. Nonglomerular hematuria (Berger’s disease, bladder tumors, renal stones, renal vascular defects, systemic intravascular hemolysis, etc.) is characterized by circular (isomorphic) erythrocytes, proteinuria, and absence of RBC casts. Glomerular hematuria is characterized by dysmorphic erythrocytes, RBC casts, and proteinuria. Urine casts also play a role in determining the cause of hematuria.^9,10

Casts are viewed in urine sediment using microscopy. Because casts are formed within the kidney as molds of the renal tubules, their presence indicates urine stasis in the background of appropriately low pH and (Tamm-Horsefall) protein concentration, as seen in glomerular disease. As many as 4 hyaline casts per low-power field may be normal, but all other casts are abnormal. A red blood cell cast is shown in the second line of Table 4. Compare these to the hyaline cast directly above, which may be seen in healthy subjects. Casts may disappear in urine with alkaline pH. This is common in urinary tract infections and old urine samples. 

If results of the microscopic examination are negative, but the clinical index of suspicion for hemoglobinuria is high, a fresh urine sample should be obtained for reevaluation. Because hematuria may be transient, if it is suspected clinically, urinalysis should be repeated 2 or 3 times at intervals of a few days. If hematuria is present in more than one specimen on different days, additional clinical investigation is warranted.

Case studies

Case 1. A 30-year-old woman went to her family physician for a routine physical exam. A urine specimen taken at that time was noted as red urine on visual exam. Physical exam and medical history were unremarkable. She mentioned that she occasionally noticed a change in urine color, but to her knowledge this did not coincide with her menstrual cycle, which was normal. She was not taking any antibiotics or medications, but she regularly took as many as 50 vitamin C tablets per day and was a vegetarian. Her urine dipstick results were positive for hemoglobin; urinalysis was unremarkable; and urine microscopy results were negative for erythrocytes. 

In this case, further questioning revealed that the patient ate many vegetables and fruits, among which were a large number of beets and blackberries. These two foods are known to cause red urine. In addition, her large doses of vitamin C (ascorbic acid), resulted in a false-positive result on urine dipstick because there was an inhibition of the peroxidase reaction. Here, her negative urinalysis and urine microscopy results were helpful in determining that this was a nonpathologic state. The clinician used this information to ask the right questions and find the answer without additional testing. 

Case 2. A 23-year-old man presented to the emergency department reporting “bloody urine” for 1 week. He first noticed this symptom the morning after running his first marathon (26 miles or 42 kilometers). He had “occasionally seen funny-colored urine” previously after running shorter distances (greater than 10 km), but this condition resolved after 2 or 3 days if he stopped running. He denied having any fever, skin rash, recent upper respiratory illness, sore throat, hypertension, history of diabetes mellitus, or family history of renal disease or deafness. He was not taking any medications and used a multivitamin. 

Physical exam showed a normotensive, afebrile man in good physical condition, without any gross abnormalities. Throat culture results were negative. Urine was a bright red color on gross evaluation. Urine dipstick findings included 4+ blood, 3+ protein, negative nitrites, and 1+ glucose. Microscopic examination demonstrated numerous dysmorphic erythrocytes and red blood cell casts. A 24-hour urine sample contained 200 mg protein/dL. Urine protein electrophoresis results were negative for a monoclonal component but positive for a selective proteinuria. Results for anti-streptolysin (ASO) titers, complement 3 (C3) level, and double-stranded DNA (dsDNA) titers were negative. A renal biopsy was performed, which demonstrated mesangial deposits of immunoglobulin A (IgA), IgG, and beta-1c globulin. These findings were consistent with Berger’s disease (IgA nephropathy). 

In this case, exercise-induced hematuria or myoglobinuria secondary to excessive trauma were the most likely diagnoses on initial presentation. The patient’s history of bloody urine after running long distances (greater than 10 km) that resolved several days later is classic for exercise-induced hematuria. However, the patient was concerned because this time the urine color did not change. His urine examination was significant for both hematuria and proteinuria, which required further investigation. Had this not been the case, a test for myoglobin could have been performed. The patient had no history of deafness or familial renal disease, thus leading away from a diagnosis of Alport’s syndrome. Goodpasture’s syndrome could be excluded because the patient was not anemic and had no lung manifestations such as hemoptysis. Negative throat culture and ASO titer results ruled out poststreptococcal glomerulonephritis. Negative complement and dsDNA titer results made the diagnosis of SLE less likely. His normal blood pressure negated the possibility of hypertensive disease, and his urine electrophoresis did not suggest myeloma. 

In this type of case, a renal biopsy is necessary to determine the cause of renal disease; and here, this indeed gave us our answer: Berger’s disease. This is the most common cause of glomerular hematuria, accounting for about 30% of cases. It is seen more commonly in males and may present initially after rigorous exercise. Fever and rash may also be present, but most patients have no associated symptoms. Gross hematuria may be intermittent, but microscopic hematuria is generally a constant finding. This disease is chronic, but most patients have no progressive renal disease. Roughly 25% will eventually develop renal insufficiency. The point to note here is that rigorous exercise alone may cause transient hematuria, but other causes that may be exacerbated by exercise must be excluded first.^1,2,8 

Case 3. A 65-year-old man with a history of urinary frequency at night (nocturia) reported to his primary care physician that his urine was pink to red in color for a few weeks. He denied any fever, chills, sore throat, or chronic diseases. He denied taking medications, vitamins, or having any recent surgical procedures. His family and personal history were negative for renal disease. He had smoked 2 packs of cigarettes per day for the past 30 years. His occupational history was significant in that he worked for 20 years in a dye factory 25 years ago, where he worked with aniline dyes, specifically beta-naphthylamine. Gross examination of the urine revealed a dark-red color. Urine dipstick results showed 4+ blood. Urine microscopy results were significant for malignant cells. Urine cytology, radiographic studies, and subsequent bladder biopsy results revealed a transitional cell carcinoma.

In this case, the patient had 2 exposures to known carcinogens—tobacco and beta-naphthylamine—that are associated with bladder cancer.^15–18 His complaint of nocturia raises suspicion of other urinary tract malignancies; but given this history, bladder carcinoma should be suspected and considered at the top of the differential list. For more than a century, aniline dyes such as beta-naphthylamine have been found to be associated with an increased risk in multiple carcinomas, especially bladder carcinoma. Often, these tumors were found on screening tests of these workers who remained asymptomatic. Debate exists on the causative agent in tobacco, which may be associated, but not causative of tumorigenesis. 

Many simple, effective tools are available to the laboratorian to evaluate urine. When hematuria is suspected, a combination of tests, including the urine dipstick, specific gravity, and urine sediment exam by microscopy can be used to narrow the spectrum of disease states in which hematuria is a symptom. Good communication with the clinician and careful evaluation to rule out toxins and drug-related urine color changes are essential. Urine, as an easily collected specimen, remains an important part of the laboratory assessment of patient health.

Rebecca M. Hsu is a Chemical Pathology Fellow and Leland B. Baskin is an Assistant Professor, Department of Pathology, Division of Clinical Biochemistry and Metabolism, University of Texas Southwestern Medical Center at Dallas, TX. Dr. Baskin is also the Associate Medical Director at Carter Blood Care, Fort Worth, TX.

References

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3. Henry JB, Lauzon RB, Schumann GB. Basic examination of urine. In: Henry JB, ed. Clinical Diagnosis and Management by Laboratory Methods. 19th ed. Philadelphia, PA: WB Saunders Co.; 1996:411–456, chap 18.

4. Peter JB, Blum RA. Use and Interpretation of Laboratory Tests in Nephrology. 2nd ed. Santa Monica, CA: Specialty Laboratories; 1997.

5. Cruz CC, Spitzer A. When you find protein or blood in the urine. Contemp Pediatr. 1998;15(9):89–109.

6. Strasinger SK. Urinalysis and Body Fluids. Philadelphia, PA: FA Davis Co.; 1994.

7. Oken DE, Schoolwerth AC. The kidneys. In: Noe DA, Rock RC, eds. Laboratory Medicine: The Selection and Interpretation of Laboratory Studies. Baltimore, MD: Williams & Wilkins; 1994:401–461, chap 21.

8. Kincaid-Smith P. Hematuria and exercise related hematuria. Br Med J. 1982;285:1595

9. Ringsrud KM, Linn JJ. Urinalysis and Body Fluids: A Color Text and Atlas. St Louis, MO: Mosby–Year Book, Inc.; 1995.

10. Graf Sr L. A Handbook of Routine Urinalysis. Philadelphia, PA: JB Lippincott Co.; 1982.

11. Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry. 3rd ed. Philadelphia, PA: WB Saunders Co.; 1999.

12. Trainor LD, Solomon HM. Detecting myoglobinuria: A low-tech analysis. Lab Med. 1997;28:569–571.

13. Blondheim SH, Margoliash E, Shafrir E. A simple test for myohemoglobinuria (myoglobinuria). JAMA. 1958;167:453–454.

14. Brown DC. Hematuria: When is it cause for alarm? Postgrad Med. 1987;82(4):112–118.

15. Vineis P, Pirastu R. Aromatic amines and cancer. Cancer Causes & Control. 1997;8(3):346–355.

16. Shinka T, Sawada Y, Morimoto S, Fujinaga T, Nakamura J, Ohkawa T. Clinical study on urothelial tumors of dye workers in Wakayama City. J Urol. 1991;146(6):1504–1507.

17. Schulte PA, Ringen K, Hemstreet GP, et al. Risk factors for bladder cancer in a cohort exposed to aromatic amines. Cancer. 1986;58(9):2156–2162.

18. Bulbulyan MA, Figgs LW, Zahm SH, et al. Cancer incidence and mortality among beta-naphthylamine and benzidine dye workers in Moscow. Int J Epidemiol. 1995;24(2):266–275.

Additional reading

Bartlett, RC, Zern DA, Ratkiewicz I, Tetreault JZ. Reagent strip screening for sediment abnormalities identified by automated microscopy in urine from patients suspected to have urinary tract disease. Arch Pathol Lab Med. 1994;118:1096–1101.

Chaplin H. Hematuria in hemoglobin S disorders. Arch Intern Med. 1980;140:1573

Cilento BG Jr, Stock JA, Kaplan GW. Hematuria in children: A practical approach. Urol Clin North Amer. 1995;22:43.

Fitzwater DS, Wyatt RJ. Hematuria. Pediatr Rev. 1994;15:102.

Gauthier B, Edelman CM Jr, Barnett HL. Asymptomatic (microscopic) hematuria. In: Nephrology and Urology for the Pediatrician. Boston, MA: Little, Brown & Co; 1982:87–91.

Kalia A, Travis LB. Hematuria, leukocyturia and cylindruria. In: Edelman CM Jr., ed. Pediatric Kidney Disease. Boston, MA: Little, Brown & Co; 1992:552–563.

Kesson AM, Talbott JM, Gyory AZ. Microscopic examination of the urine. Lancet. 1978;809.

Numazaki Y, Kumasaka T, Yano N, et al. Further study on acute hemorrhagic cystitis due to adenovirus type II. N Engl J Med. 1973;289:344.

Yadin O. Hematuria in children. Pediatr Ann. 1994;23:474.