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This focus topic is the second of a two part series on urine specimen collection. The second article covers sources of preanalytical artifact arising during urine collection, handling and transportation. As we stated in part one, urine has a long history as a specimen for analysis in clinical laboratories. After blood, urine is the most commonly used specimen for diagnostic testing, monitoring of disease status and detection of drugs. Urine testing using both automated and traditional manual methods is growing rapidly1. As for all clinical laboratory specimens, preanalytical error in urine specimens is often difficult to detect. Because of this, it is important for laboratories to have processes in place to ensure compliance with best practice in specimen collection, handling and transport.
The use of good quality collection devices and containers is an important part of the provision of consistently high quality urine specimens. The Clinical and Laboratory Standards Institute (CLSI) recommends the use of appropriate urine collection tubes, leak-proof containers, pipettes and standardized microscope slides with calibrated volumes for analysis of urine sediment2. Tubes manufactured using clear plastic and with conical bases are recommended for microscopic analysis of urine sediment.
Consideration should be given as to whether preserved or unpreserved specimens are used. Proliferation of bacteria can be a major preanalytical issue with unpreserved specimens3.
If a specimen has been refrigerated for storage or transportation, it should be allowed to equilibrate to room temperature and be well mixed prior to any type of analysis.
Because many medications can interfere with testing of analytes in urine, it may be necessary to discontinue a particular medication for a period of time to enable collection of a ‘drug-free’ specimen.
Two common factors affecting urine dipstick and other testing are color and clarity of the specimen.
A number of substances can interfere with dipstick tests. Examples of these are provided below. Please note that the effect of these substances will vary according to the type of test strip used. Commonly performed dipstick tests include:
- Specific Gravity (density)
- Leukocyte Esterase
The value of random urine testing for chemistry analytes is limited because of the lack of accumulation of these at any given time in the bladder. Timed specimens provide the most valuable information for the urinary excretion or clearance of specific analytes. It should be noted however that one of the most significant sources of preanalytical errors in quantitative urine analysis is the failure to collect ALL urine passed during the stated collection period.
Many of the preanalytical variables described in this article for urine dipstick testing apply equally to urinalysis conducted using more sophisticated analytical procedures in the laboratory. Analytes on the following list may be associated with specific sources of preanalytical artifact:
- Total Protein
- Uric Acid
- 5-hydroxyindoleacetic acid (5-HIAA)
- Catecholamines and metanephrines
- Drugs of Abuse (DOA)
- Amino Acids
- Organic Acids
Urine Culture and Antibiotic Susceptibility Testing
Preanalytical variables that can affect microbiological culture and antibiotic susceptibility testing include:
- Contaminated collection containers (e.g. bacterial contamination - from hands, skin, clothing, leaking specimen containers; contamination with antiseptics – from hands, traces of bacteriostatic or bacteriocidal agents in the container)
- Type of specimen. A midstream clean catch specimen is less likely to contain bacterial contaminants than a random urine specimen. The bacterial contamination rate for females is double the rate for males6. Contamination may be defined as more than 10,000 CFU/mL of two or more organisms but each laboratoryshould define its own criteria - based on the collection and transport methods in use.
- Delays in specimen transportation. Specimens that cannot be transported immediately to the laboratory2 (or unable to be refrigerated if immediate transport is not possible2), or do not have a bacteriostatic preservative may undergo bacterial overgrowth leading to falsely elevated colony counts.
- Antibiotics. False negative or reduced bacterial growth may be seen in specimens from patients receiving antibiotic medication prior to urine collection.
- Frost and Sullivan Research Service. Global in vitro diagnostic market outlook. San Antonio (TX): Frost and Sullivan; 2005.
- CLSI. Urinalysis; Approved Guideline – Third Edition. CLSI Document GP16-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2009.
- Journal of Clinical Microbiology, Evaluation of Liquid and Lyophilized Preservatives for Urine Culture. 1983 (Oct): 912-916.
- Berman LB. Urine in technicolor. JAMA. 1974 May 6;228(6):753
- Fleisher DS. Urine of abnormal color. Pediatrics. 1968 Sep;42(3):545-6.
- Raymond JR, Yarger WE. Abnormal urine color: differential diagnosis. South Med J. 1988 Jul;81(7):837-41.
- Hsu RM, Baskin LB. Laboratory evaluation of discolored urine. When is it hematuria? MLO Med Lab Obs. 2000 Jul;32(7):44-52
- Slawson M. Thirty-three drugs that discolor urine and/or stools. RN. 1980 Jan;43(1):40-1.
- Saran R, Abdullah S, Goel S, Nolph KD, Terry BE. An unusual cause of pink urine. Nephrol Dial Transplant. 1998 Jun;13(6):1579-80
- Masuda A, Hirota K, Satone T, Ito Y. Pink urine during propofol anesthesia. Anesth Analg. 1996 Sep;83(3):666-7.
- RosenbergJW. Phenytoin and red urine. JAMA. 1983 Oct 14;250(14):1842-3.
- Lord RC. Orange urine. Postgrad Med J. 1999 Feb;75(880):109-10.
- Blakey SA, Hixson-Wallace JA. Clinical significance of rare and benign side effects: propofol and green urine. Pharmacotherapy. 2000 Sep;20(9):1120-2.
- Drummond KN, Michael AF, Ulstrom RA, Good RA. The blue diaper syndrome: familial hypercalcemia with nephrocalcinosis and indicanuria. A new familial disease, with definition of the metabolic abnormality. Am J Med 1964; 37: 928Ð948.
- Noll WW, Glass DD. Causes of dark urine. JAMA. 1980 Jun 20;243(23):2398.
- Baker MD, Baldassano RN. Povidone iodine as a cause of factitious hematuria and abnormal urine coloration in the pediatric emergency department. Pediatr Emerg Care. 1989 Dec;5(4):240-1.
- Jimbow K, Lee SK, King MG, Hara H, Chen H, Dakour J, Marusyk H. Melanin pigments and melanosomal proteins as differentiation markers unique to normal and neoplastic melanocytes. J Invest Dermatol. 1993 Mar;100(3):259S-268S.
- Adonis-Koffy L, Gonzales E, Nathanson S, Spodek C, Bensman A. Alcaptonuria: a rare cause of urine discoloration. Report of a case in a newborn. Arch Pediatr. 2000 Aug;7(8):844-6.
- Rich MW. Porphyria cutanea tarda. Don't forget to look at the urine. Postgrad Med. 1999 Apr;105(4):208-10, 213-4
- Graff L. A Handbook of Routine Urinalysis. Philadelphia: Lippincott, Williams and Wilkins; 1982.
- Ringsrud KM and Linne. Urinalysis and Body Fluids: A Color Text and Atlas. Mosby; 1995: 45.
- Skobe C. Preanalytical variables in urine testing. BD LabNotes 2006; 16(3):1-7
- WHO Guidelines on Standard Operating Procedures for Clinical Chemistry: 2006. Blood Safety and Clinical Technology, Section D – Urinalysis.
- Schwarzhoff R, Cody JT. The effects of adultering agents on FPIA analysis of urine for drugs of abuse. J Anal Toxicol 17, 14-17 (1993).
- Marshall T and Williams KM. Total Protein Determination in Urine: Aminoglycoside Interference. Clin Chem 2000;49(1):202-203.
- Thomsen HS and Morcos SK. Contrast media and the kidney: European Society of Urogenital Radiology (USUR) Guidelines. British Journal of Radiology 2003; 76:513-518
- Stein HB and Hasan A. and Fox Ih. Ascorbic acid-induced uricosuria. A consequence of megavitamin therapy. Ann Intern Med;84(4):385-8.
The colour of urine, which is normally colourless or one of the various shades of yellow, can be altered by medications, vitamins, dyes, or diet. Some abnormal urine colours and their possible causes are:
- Red – due to the presence of blood (or hemoglobin), myoglobin (as a result of break down of muscle cells),medications (phenytoin, phenothiazines),artificial colours in some laxatives, beetroot, black berries and rhubarb due to the presence of anthrocyanin.6-11
- Orange - phenazopyridine (Pyridium) and ethoxazene (Serenium) which are used as urinary tract anaesthetics to diminish dysuria. Rifampin, phenacetin, sulfasalazine, vitamin C, riboflavin, and carrots will also turn urine dark yellow to orange.8,12
- Black – due to the presence of melanin in patients with advanced melanoma, presence of homogentisic acid in alcaptonia, a rare hereditary disease.18
- Brown – due to the presence of bilirubin (e.g. in patients with obstructive jaundice), contamination with povidone-iodine (Betadine™) solution or douche,medications causing brown or brown-black urine include:chloraquine and primaquine, furazolidone, metronidazole, nitrofurantoin, cascara/senna laxatives, methocarbamol, and sorbitol.8, 15-19
- Green – resulting from the use of some medications (e.g. chlorophyll in some mouthwash products), propofol (an anesthetic).13
- Green/blue – seen with Pseudomonas infection, rinsapin (an antibiotic used to treat staph infection), triamterene (a mild diuretic), and methylene blue (a component in several medications), Amitriptyline, indomethacin, resorcinol, cimetidine, phenergan, and some multivitamins preparations also lend a blue-green tint to the urine.4,5,8,13,14
- Yellow – excessive yellow colouration can occur with intake of high doses of riboflavin vitamin supplement 22.
These abnormal urine colours can affect dipstick results by causing misinterpretation of colour panels on the test strips (manual and automated assessments).
Normal urine is clear. Urine clarity may be affected by some handling and transportation conditions. If a urine specimen is unpreserved, it can become cloudy from bacterial overgrowth. Refrigeration may lead to precipitation of amorphous urates or phosphates resulting in cloudiness. This will generally disappear when the specimen is brought to room temperature. The collection time and storage conditions of the specimen should be reviewed to determine if cloudiness may have been caused by storage conditions. Other causes of a lack of clarity include the presence of mucus, crystals, leukocytes, epithelial cells, fat globules and contamination with talcum powder. As for abnormal colouration, a lack of clarity may lead to test result inaccuracies.
This reflects the amount of dissolved substances in the urine and serves as a measure of renal function. Normal urine has a specific gravity within the range 1.001–1.035.
Preanalytical variables affecting urine specific gravity include dehydration (e.g. due to excessive sweating, diarrhoea, and prescription of some antibiotics) because of the distribution of dissolved solids in reduced volumes of urine. Radio-opaque dyes can also cause an increase in urine density. Conversely, increased fluid consumptionor intake of diuretics can reduce urine specific gravity because of larger volumes of urine20.
Normal urine pH falls within the range 5 to 7. pH values provide a useful guide for subsequent microscopic examination. For example, certain crystals exist in either an acidic or alkaline environment. Uric acid and calcium oxalate crystals are more likely to be seen in acidic urine whilst calcium carbonate and magnesium phosphate crystals are more likely to be seen in alkaline urine. Casts may dissolve in dilute urine. Cells are more susceptible to lysis in dilute urine.
A number of preanalytical variables can affect urine pH. Bacterial overgrowth in a specimen standing at room temperature will often lead to a higher pH due to the conversion of urea to ammonia21. Diets with a high content of vegetables and citrus fruits may produce an alkaline pH20.Urine pH will also be alkaline in respiratory alkalosis (hyperventilation) and in metabolic alkalosis (e.g. severe vomiting)20. Post prandial urine specimens are generally more alkaline than those collected at other times due to the excretion of acid into the stomach after eating – so called ‘alkaline tide’20. Urine specimens with lower pH levels may be seen in uncontrolled diabetes (ketoacidosis). Starvation and diarrhoea can also lead to the production of acidic urine20.
This is a very important analyte in urine. Protein is measured to assess the function of the kidneys. Normal urine contains no more than trace amounts of protein. Apart from renal insufficiency, total protein in urine may be increased following heavy exercise, dehydration, very high protein intake and emotional stress20,22. Contamination with disinfectants(e.g. quarternary ammonium compounds) is one source of preanalytical error in protein determination by dipstick in urine specimens. This contamination typically leads to false positive protein results20. False positives may also be seen in strongly alkaline urines and in those contaminated with vaginal or urethral secretions20. Dilute specimens may also produce false negative protein results20.
Urine dipsticks are used to detect intact red blood cells, free hemoglobin, and myoglobin. Normal urine specimens test negative for blood. False positives can be caused by the presence of hypochlorite (bleach)20 and consumption of colored medications and foods containing dyes22. Microbial peroxidase can also cause false positive reactions20. The introduction of blood during the specimen collection procedure (e.g. menstrual blood) is another source of preanalytical error. Another source of preanalytical error is ascorbic acid which can lead to false negative results20.
This is a commonly available test on urine dipsticks. Normal urine is negative for nitrite, the presence of which is indicative of bacterial infection. Preanalytical variables include bacterial overgrowth in the specimen which leads to conversion of nitrate to nitrite in specimens that have not been stored correctly20. False negative results for nitrite may be seen when ascorbic acid is present (at levels as low as 50 mg/L), when the urine contains abnormally high levels of urobilinogen or if the pH is below 6.020. Note thatthe conversion of nitrate to nitrite takes approximately four hours in the bladder. If this has not occurred, results could be negative for the presence of nitrite. This can be seen in randomly collected specimens.
This is an indicator for the presence of white blood cells and thus is also a marker for infection. Normal urine specimens are negative for leukocyte esterase. False positives may be obtained when collection containers have been contaminated with hypochlorite (bleach) or detergents22. Other preanalytical factors leading to false positive results include formalin (when used as a preservative) and vaginal discharge22. High specific gravity, some antibiotics (e.g. tetracyclines) and large amounts of glucose or ascorbic acid can also cause false negative leukocyte esterase results20.
This is used to detect diabetes mellitus. Normal urine contains nil or trace amounts of glucose. These trace levels are below levels of sensitivity for commonly used reagent strips. However, glucose may be present in the urine of some normal individuals who have a low glucose renal threshold. False positive glucose results may be seen when collection devices have been contaminated with formalin20. Nalidixic acid and cephalosporins may also cause false positive results20. High concentrations of ascorbic acid have been implicated as causing false positive results but this has been the subject of debate20. Over time at room temperature, glucose levels in urine may decrease due to glycolysis mediated by bacteria22.
These are a by-product of fat breakdown and are not present in normal urine. They are associated with decreased intake of carbohydrates (starvation), decreased utilisation of carbohydrates (diabetes mellitus), eclampsia, severe vomiting and diarrhoea20. One of the ketones is acetone which is very volatile. Because of this, urine specimens must be tested immediately after collection or refrigerated in a sealed container to prevent loss of acetone to the atmosphere20. Weak false positive reactions may be seen in specimens containing L-dopa or phenylpyruvic acid23.
Elevated in the blood of patients with abnormal liver function, bilirubin is not present in normal urine. Coloured medications (those in the yellow, orange, and red spectrum) may cause false positive results with test strips22. Falsely elevated test results may be seen in specimens from patients receiving large doses of chlorpromazine20. Metabolites of drugs such as phenazopyridine which produce a red color at low pH may also cause false positive results20. High levels of ascorbic acid may cause false negative results, as can the presence of nitrite in high concentrations20. Exposure to light causes bilirubin to degrade over time, leading to falsely reduced or false negative results. It is recommended therefore that specimens submitted for bilirubin measurements be protected from light or collected in an amber container.
Urobilinogen levels are elevated in urine of patients with abnormal liver function. Normal urine contains less than 10 Ehrlich units/L. As for bilirubin, urobilinogen is also light sensitive22. Nitrite concentrations greater than 50mg/L and formalin concentrations above 2g/L may cause false negative results20.
Creatinine levels may be falsely increased by ascorbic acid in urine specimens24
Calcium levels may increase in patients receiving antacids, anticonvulsants, and some diuretics (e.g. loop diuretics such as Frusemide) while adrenocorticosteroids, thiazide diuretics and oral contraceptives may cause decreased levels22.
Total protein results may be falsely elevated in the presence of some antibiotics25. X-ray contrast media may also cause falsely elevated test results26.
Microalbumin is increased by dehydration or strenuous exercise22.
Uric acid levels in urine have been reported to be increased by high levels of ascorbic acid27. Other sources of interference for uric acid analysis include alcohol, anti-inflammatory drugs, salicylate, and warfarin22.
Amylase test results can be falsely elevated by the presence of aspirin, corticosteroids, codeine, and oral contraceptives in the specimen22.
5-HIAA levels in urine are influenced by many types of foods, such as plums, pineapples, walnuts, avocados, tomatoes, bananas, and eggplant. It is recommended that they should not be consumed for three days prior to testing. 5-HIAA test results can also be falsely elevated by some cough syrup formulations22. Levels can be falsely decreased by heparin, methyldopa, and tricyclic antidepressants22.
Catecholaminescomprise of dopamine , noradrenaline (norepinephrine), and adrenaline (epinephrine). Metanephrines are metabolites of catecholamines which comprise of metanephrine and normetanephrine.Analysis of these hormones/metabolites ishelpful in diagnosis or ruling out the presence of pheochromocytoma or other neuroendocrine tumor. Measurement of their levels in urine is influenced by chocolate, cocoa, coffee, tea, bananas, and other vanillin compounds in the diet. Such interference occurs only with colorimetric assay methods. HPLC methods are not subject to this interference. Catecholamine and metanephrine levels are also affected by emotional and physical stress and vigorous exercise and can be increased by medications including lithium, insulin, tetracycline, and nitroglycerin. Levels of catecholamines may be decreased in patients receiving salicylates and imipramine22.
Drugs of abuse (DOA) testing on urine specimens present additional challenges due to the potential for tampering with these specimens. Substances commonly added to DOA specimens include water, hypochlorite (bleach), vinegar and poppy seeds (to produce very high opiate levels to mask the presence of other drugs). Commercially produced ‘adulterants’ are also available on the ‘black market’.
Simultaneous quantitative analysis of amino acids and creatinine is necessary for the diagnosis of inherited metabolic diseases due to abnormal metabolism of amino acids. Random urine must be collected with no preservative at the time when the patient shows acute symptoms and has not received any medical intervention, such as dietary restriction, administration of intravenous fluids, vitamins and/or medications. Urine specimen must be sent to the laboratory on ice within one half hour of collection.
Analysis of organic acids is required for the diagnosis of inherited metabolic diseases which produce abnormal quantity and/or variety of organic acids. Random urine must be collected with no preservative at the time when the patient shows acute symptoms and has not received any medical intervention, such as dietary restriction, administration of intravenous fluids, vitamins and/or medications. Urine specimen must be sent to the laboratory on ice within one half hour of collection.
Last modified 15th November 2012