Best Practices for Achieving Optimal Specimen Quality and Accurate Test Results
Catherine Skobe, MT (ASCP)
There are many preanalytical variables that can introduce error into laboratory test results. When unexpected results are seen in urine measurements, they should be interpreted in the context of other analytes and clinical results that measure similar aspects of renal function. An example is blood and bacteria testing in routine urinalysis. A false-positive result for blood can be obtained when a specimen contains infection-causing bacteria. The microbial peroxidase activity can cause a false-positive reaction for blood. In this case, microscopic analysis will verify the presence of bacteria and possibly the lack of red blood cells. This will confirm that the response for blood on the reagent strip is inaccurate. Another example is the effect of urine colour on urinalysis reagent test pads. Abnormally coloured urine due to medications, food dyes, or vitamins can alter the colour change on dipstick test pads for tests other than colour. This can be ruled out by doing a visual exam of the urine specimen prior to performing the dipstick. For this reason, the College of American Pathologists (CAP) recommends that laboratories have a procedure for the correlation of microscopic and macroscopic results. In general, collection time, transport, and storage conditions can be examined to determine the causes of error. There are some basic guidelines that should be followed for urinalysis and the storage and handling of urinalysis dipsticks. By following these precautions, inaccurate results can be minimized.
Preanalytical Variables Colour 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. If an unusual colour is detected for the urine specimen, one of these conditions could be the cause. Once the cause is determined, it should be noted in the laboratory results. Some abnormal urine colours and their possible causes are:
These abnormal urine colours can affect other dipstick results by causing a colourimetric reaction that may be misinterpreted by the instrument and give incorrect results. It is important that the tube be of a clear material when determining the colour of the urine. Using good lighting and a white background helps to ensure the colour is being read accurately and consistently. Colour descriptions should be standardized. Clarity Another visual measurement is clarity. (A normal urine specimen is typically clear.) Urine clarity can be related to the handling conditions of the specimen. If a urine specimen is old and unpreserved, it can become cloudy from bacterial overgrowth. In turn, if a specimen has been stored in a refrigerator, amorphous urates or phosphates can cause temporary cloudiness (which dissolve when the specimen is brought to room temperature). Amorphous urates are observed in acidic urines and amorphous phosphates are found in alkaline urine. The collection time and storage conditions of the specimen should be reviewed to determine if cloudiness may be caused by storage conditions. Other causes of cloudy samples include talcum powder, mucus, crystals, leukocytes, epithelial cells, and fat. Clear tubes are best for examining urine clarity. Result reporting should be standardized, with clear, hazy, cloudy, and turbid being the most commonly used descriptors. Specific gravity, or the amount of dissolved particles in a solution, is another measurement performed on urine. Specific gravity is affected by the number, amount, and weight of solutes in the specimen. It serves as a measure of the kidney's ability to dilute and concentrate urine. A normal random urine has a specific gravity range of 1.001-1.035. Dehydration, sweating, diarrhea, radiopaque dyes, and antibiotics can cause high results because the ratio of dissolved particles in low volumes of solute will be elevated. A high fluid intake or consumption of diuretics can cause low measurements because of the low quantity of dissolved particles in a large volume of solute. The pH test indicates whether a specimen is acidic (pH <7) or alkaline (pH >7). A normal urine pH ranges from 5-7 and is a useful tool for the laboratorian, often predicting what may be seen in subsequent microscopic examination. Certain crystals exist in either an acidic or alkaline environment. Some examples of these are uric acid or calcium oxalate crystals in acidic urine and calcium carbonate or magnesium phosphate in alkaline urine. Dilute and alkaline urines can dissolve casts and cells. Bacterial overgrowth in a specimen standing at room temperature will produce a higher pH due to the conversion of urea into ammonia.1 Diets high in vegetables, citrus fruits, and dairy produce an alkaline pH. Lower pH levels may be seen in uncontrolled diabetes or may reflect a diet high in meat or cranberries. Starvation and diarrhea can produce a more acidic urine. Lastly, mishandling the reagent strips by allowing runover from the protein reagent pad can cause false negative pH results. Protein is a very important analyte measured in urine because it monitors the function of the kidneys. A normal urine specimen should not contain more than trace amounts of protein. Most reagent pads primarily measure albumin and, to a lesser extent, other proteins. Residue of disinfectants in contaminated urine containers can cause false-positive protein results. Similar false positives may be seen after strenuous exercise and in highly alkaline urines. Urine specimens with a high specific gravity can produce trace results. Dilute specimens, fever, mental stress, mucus, and exposure to extreme heat or cold can produce falsenegative protein results. Blood is another measurement on the urine dipstick, detecting intact red blood cells, free hemoglobin, and myoglobin. A normal urine specimen is usually negative for blood. False positives can be caused by the presence of chlorine bleach, consumption of colourred medications by the patient, or a microbial peroxidase reaction from bacterial presence. False-positive-causing contaminants can also be introduced during time of collection, such as in specimens submitted by women during menstruation. High levels of a reducing substance such as ascorbic acid at >25 mg/dL can produce falsenegative results. False-negative blood results can also occur when formalin is used as a preservative, when there is>10 mg/dL of nitrite, or when mixing is inadequate and red blood cells have settled in the tube. Nitrite is also a test on urine dipsticks. A normal urine specimen is negative for nitrite. Some of the ways in which falsepositive results can occur include bacterial overgrowth in specimens that have not been stored properly, coloured medications, and dyes. False-negative results can be seen with high levels of ascorbic acid, >25 mg/dL, or when dietary nitrate is missing due to starvation, IV feeding, or fasting. The conversion of nitrate to nitrite takes about 4 hours in the bladder. If this hasn't occurred, results could be negative for the presence of nitrite. This can be seen in randomly collected specimens. Leukocyte esterase is an indicator of white blood cells. It is typically negative in normal specimens. False positives can occur when collection containers have been contaminated with chlorine bleach or other oxidizing detergents. Other influences that can produce false-positive results include formalin as a preservative and vaginal discharge. High specific gravity, some antibiotics, such as tetracycline, and large amounts of glucose or ascorbic acid can cause false negative leukocyte esterase results. Glucose, another urine dipstick test, is mainly used to monitor diabetes. Normal urines are negative for glucose. Some normal specimens have small amounts of glucose, that are below levels of sensitivity for the reagent strip. Just like leukocyte esterase, false-positive glucose results are achieved when collection devices have been exposed to chlorine bleach or detergents. Improper storage of reagent strips, when exposed to air, have been noted to produce false-positive results. Ascorbic acid, >50 mg/dL, is again a culprit in causing false-negative glucose results. Over time at room temperature, glucose will decrease due to glycolysis from bacteria. Tetracycline has been determined to cause false-negative glucose results, and refrigerated specimens that were not allowed to reach room temperature can produce false-negative results because the enzymatic reaction is affected. Ketone bodies are a by-product of fat breakdown. Normal urines are negative for ketones. Increased ketones can be due to starvation or alcoholism. Also strenuous exercise, fever, fasting, vomiting, and high protein diets can cause high ketone values. Urine specimens with a high specific gravity and low pH have been known to cause trace ketone results. Acetone, one by-product of fat breakdown, evaporates rapidly if the uncovered specimen is left standing at room temperature. Specimens should, therefore, be tested immediately for ketones or refrigerated in a closed container.2 Some urinalysis preservatives can maintain ketone levels. It is important to check the manufacturer's claims. Bilirubin measures liver function and is usually negative in normal urine specimens. Coloured medications, those in the yellow, orange, and red spectrum, can cause false-positive results. Ascorbic acid, >25 mg/dL, causes false negative results, as can specimens with high nitrite concentrations. Exposure to light causes bilirubin to degrade over time, leading to false-negative results. It is recommended that specimens submitted for bilirubin measurements be kept in a dark place or collected in an amber container. Urobilinogen is another measurement of liver function. A normal urobilinogen result is <1 Ehrlich unit/dL. The same factors that affect bilirubin (exposure to light and coloured medications) also affect urobilinogen. In addition, formalin, which is sometimes used as a preservative, will cause false-negative urobilinogen results, as can ascorbic acid or nitrite. Standardization when processing urine specimens for microscopic sediment analysis has become a very important guideline as recommended by the Clinical and Laboratory Standards Institute (CLSI, formerly known as NCCLS). This includes the use of engineered tubes, pipettes, and standardized calibrated slides. The best types of tubes for microscopic sediment analysis are clear, plastic tubes with conical bottoms. A cap or lid and volume gradations are valuable features. CLSI does not support the use of glass slides and cover slips due to the lack of sample volume standardization. The chambers on the specially designed slides are calibrated for a specific urine sediment volume that ensures standardization.Urine Culture and Sensitivity The microbiology lab also conducts clinical testing on urine. Preanalytical variables that could affect culture and sensitivity testing include:
Urine Chemistry
References
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