Kirandeep Panesar,
BPharm (Hons), MRPharmS, RPh, CPh
Freelance Medical Writer
Orlando, Florida,
Consultant Pharmacist
Acacia Pharmacy,
Nairobi, Kenya
FACULTY DISCLOSURE STATEMENTS:
Kirandeep Panesar has no actual or potential conflict of interest in relation to this activity.
U.S. Pharmacist does not view the existence of relationships as an implication of bias or that the value of the material is decreased. The content of the activity was planned to be balanced, objective, and scientifically rigorous. Occasionally, authors may express opinions that represent their own viewpoint. Conclusions drawn by participants should be derived from objective analysis of scientific data.
The evolving role of pharmacists as providers of health and health care information encompasses both in-pharmacy clinical testing and the provision of counseling on tests carried out in the more traditional laboratory setting. Clinical laboratory tests can be used to confirm or support a diagnosis, assess the severity of a disease (e.g., MRI for tumor size), monitor the response to treatment, monitor appropriate drug dosing, and help prevent toxic side effects and interactions.1 To facilitate the role of pharmacists in health promotion, a basic knowledge of clinical testing is useful for both institutional and retail pharmacists. This will assist pharmacists when counseling patients on preparing for laboratory procedures and receiving test results; adjusting drug therapy based on test results; providing in-pharmacy testing to promote patient health; and liaising with other health professionals to optimize patient therapy.
Urine testing provides a broad spectrum of information to health care providers and offers a noninvasive and therefore pain-free option to diagnostic testing for a number of conditions. Urinalysis has been used in diagnostic medicine for thousands of years, dating as far back as ancient Egypt, where there are descriptions of polyuria. “In the 7th century AD, Protosharis suggested that it was important to look at the color of urine and by 1674 Thomas Willis, a professor at Oxford University, recorded that the urine of his patients with diabetes tasted sweet.”2 However, it is only with the introduction of the microscope in the 19th century that there have been major advances in this field. Dipstick testing, the application of modern chemical and microscopic techniques to constituent analysis, automation, and, most recently, monoclonal antibody and recombinant gene technology, have enhanced and improved urine examination.3
Today, urinalysis is a popular test in medical diagnosis and is frequently ordered with hospital admissions and regular checkups since it is relatively simple, inexpensive, and pain free. It may specifically be ordered in patients complaining of lower back pain, abdominal pain, and problems with urination or in pregnancy. Most importantly, testing urine samples may reveal disease conditions that would otherwise go unnoticed. For example, it has been shown that rising thyroid-stimulating hormone levels may be due to a rare condition called nephrotic syndrome and not to poor compliance with thyroxine therapy. This condition can be suspected with dipstick urinalysis and confirmed with biochemical testing and a renal biopsy.4
The test itself can be broken down into three components: visual examination (macroscopic analysis), dip-and-read tests, and microscopic analysis. There are a large number of dip-and-read tests now available for OTC purchase, and pharmacists will often be asked to provide instructions on the use of these kits. Additionally, pharmacists may be required to counsel patients on collecting urine samples, interpreting test results, and adjusting drug therapy based on these results.
COLLECTING URINE FOR TESTING
Obtaining a good urine sample is a tricky procedure, since the patient provides a majority of the samples. Consequently, patients need to be clear on how and when to collect the sample. Clean-catch of midstream urine collection is suitable for most test procedures. Some tests may require timed urine collection such as the 24-hour urine collection. In hospitals, urine samples from inpatients are frequently collected by catheterization. Recently, the accuracy of the 24-hour sample, a time-consuming and inconvenient procedure, has been questioned for testing proteinuria. Accordingly, the National Kidney Foundation recommends the use of spot urine samples that correct for creatinine concentration over the 24-hour sample.5
Patients who are required to collect their own samples should ideally be provided with a container specifically designed for urine collection, free of any chemicals that may affect the test results. When testing babies, the parent or caregiver should be given a urine collection bag. Specific advice for patients collecting urine is given in TABLE 1.
Urine samples should be tested immediately after collection to prevent false results. If immediate testing is not possible, the sample should be refrigerated and allowed to return to room temperature before testing. Samples that are stored incorrectly may give inaccurate results owing to reduced clarity because of crystallization of solutes; increased alkalinity (pH) due to the loss of CO2; loss of ketone bodies and bilirubin (if present); dissolution of cells and casts; and overgrowth of contaminating microorganisms.
VISUAL EXAMINATION (MACROSCOPIC ANALYSIS)
Normal urine is a clear, pale yellow-colored fluid that has a slight odor of urea and is slightly acidic; any changes in the color, clarity, and smell of a urine sample can signal potential problems that may require further investigation. Dehydration results in an increase in the concentration of urochrome, the chemical that gives urine its distinct color, causing a darkening of the urine to an orange hue. Often, changes in urine color are a result of certain foods or drugs, and pharmacists should essentially rule out this possibility (TABLE 2). Blue urine is distinctive of the rare, inherited metabolic disorder termed the blue diaper syndrome. It is caused by incomplete breakdown and intestinal absorption of tryptophan in children.
The odor of urine may also provide some direction toward a diagnosis. Patients who have diabetes mellitus pass sweet, fruity-smelling urine caused by the excretion of acetone, and after prolonged bladder retention, alkaline fermentation causes an ammoniacal odor.6 The urine of anorexic patients often smells like pear drops, and patients with urinary tract infections (UTIs) often pass urine with a pungent smell. Gastrointestinal-bladder fistulas in which feces may be deposited in the bladder commonly result in urine with a fecal odor; the formation of sulfur compounds in cystine decomposition disorders causes urine to have a sulfuric smell.6
DIP-AND-READ TESTING
How to Use the Test Strips
Dipsticks are very simple and convenient to use, but results can be easily misinterpreted since they are semi-quantitative. Semiquantitative analysis reports results as a range within which the actual result may lie, using a color chart or other qualitative reading. Reagent strips can be used to test pH, urine specific gravity (USG), UTIs, proteins, glucose, ketone bodies, bilirubin, and urobilinogen, and to confirm pregnancy and the presence of certain drugs (TABLE 3). However, the reliability of this method of testing is still controversial since the results may be highly inaccurate.
When using a dipstick, the urine specimen should be collected in a sample bottle. Then the test area of the strip should be completely immersed in the urine sample and removed immediately. Shake off any excess urine by gently tapping the strip against the side of the specimen container. The strip should be left to react for the prescribed time. Finally, compare the color of the test area to the chart provided.7
Storage of Test Strips
Test strips should be stored at a constant temperature between 15°C and 30°C in a tightly closed container. The provided desiccant should not be removed from the container. Bayer Multistix 10 SG test strips exposed to the air for prolonged times have been shown to give false-positive results for glucose and false-negative results for microscopic hematuria (TABLE 4).8 Furthermore, Gallagher et al found that out of nine reagents tested in the Chemstrip-9 dipstick (leukocyte esterase [LE], pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, and nitrites), only the nitrite portion of the exposed dipsticks showed a rapid cumulative loss of specificity over time.9 All other tests demonstrated reproducible results after exposure to room temperature and humidity for 1 to 15 days.9 Therefore, even though manufacturers indicate that the strips should be stored in a tightly closed container and the desiccant should not be removed, all reactants may not lose their activity if exposed to air or humidity. However, it is a good practice to follow the label guidelines, and patients should be similarly advised. Discard any strips past their expiration date or any unused strips after 6 months of first opening.
Uses of Dip-and-Read Testing
Urinary Tract Infections: UTIs are characterized by an increase in the enzyme LE, nitrite, and protein levels. LE is a component of white blood cells that is normally found with bacteria, and nitrite levels rise in urinary infections since the pathogens reduce urine nitrate to nitrite.12 Most dipsticks test for at least two of these chemicals depending on the brand used.
Numerous studies, meta-analyses, and reviews have been conducted to determine the accuracy of dip-stick testing for UTIs and compare the reliability of reagent strips to culture assays, but there remains a discrepancy over the results. Yuen et al reviewed the use of leukocyte esterase dipstick testing in children and concluded that it should not be used as a replacement for microscopic examination.13 Conversely, a recent meta-analysis showed that a true negative can be concluded if both nitrites and leukocyte esterase test negative with a dipstick test.14 Moreover, Gorelick and Shaw demonstrated that the results of a dipstick analysis for both nitrite and leukocyte esterase are comparable to those of a Gram stain test and “superior to a microscopic analysis for pyuria.”15
However, in patients with a high risk of contracting a UTI, it has been shown that a negative urine dip-stick test does not rule out the possibility of an infection.16 More specifically in the long-term care setting, high rates of false-negative results for LE and nitrite with dipstick tests have been demonstrated.12 With an aim to overcoming this discrepancy, Mariani et al have proposed a protocol to determine which results of screening urine specimens that are positive on a dip-stick culture would be confirmed by a properly performed microscopic urinalysis.17
In an attempt to contain costs and save time by eliminating the urine culture step, the use of immunochromatography strips using antibodies to detect infectious agents has been examined.18 Even though immunochromatography has been found to be useful in urinalysis, this form of testing is yet to be fully developed. The newer dipslides offer a convenient way to perform a culture assay. They consist of a sterile paddle that is coated with agar on both sides, ready to use in a clear plastic tube.
Urinary pH: Health care professionals may use the urinary pH to select drug therapy for UTIs. The effectiveness of some antibiotics used to treat UTIs is affected by the urinary pH. Methenamine, for example, works only in an acidic environment, whereas streptomycin and neomycin work better in an alkaline environment.7 Urinary pH may also be used to reduce the risk of kidney stones. Uric acid and calcium oxalate stones form well in acidic urine, whereas alkaline urine is optimal for the formation of calcium carbonate and calcium phosphate stones.
Desai and Assimos indicated that determining uri-nary pH is an important part of the initial evaluation of certain patients with nephrolithiasis.19 Urinary pH manipulation therapy may be used to treat patients with cystine and uric acid stones, which results in the dissolution of uric acid stones. Monitoring the pH of patients receiving therapy for hypocitraturia is necessary to ensure that the pH does not go above 7.0, since overalkalinization can cause the formation of calcium phosphate stones.
Urinary pH is normally acidic, ranging from 4.5 to 8.0, but it is not as well regulated as blood pH due the absence of buffers in the urine. An elevated (alkaline) pH may be the result of:
- Aged urine specimen; the loss of CO2 causes increased alkalinity.
- Systemic alkalosis; this can be further confirmed by testing serum pH, which is a better indicator.
- Urine left at room temperature; Cook et al demonstrated that urine stored at room temperature has a pH of >9.0 and that the pH of samples stored below -20°C was relatively stable.20
- Infection; bacteria break down urea to ammonia that combines with hydrogen ions and raises pH.
- Certain medications (e.g., antiepileptics that have been shown to cause an increased tendency to form alkaline urine as the number of coadministered antiepileptics increases).21
- Diet high in citrate.
- Magnesium-ammonium phosphate crystals.
Acidic urine may be the result of ingestion of proteins or acidic foods. Urinary pH can also be used to detect renal tubular acidosis. In this condition, the patient is unable to acidify urine to a pH of less than 5.5 following the administration of an acid load and an overnight fast.
Therefore, it is important for pharmacists to emphasize that urine that is not tested immediately should be stored in a refrigerator and to encourage patients to take the sample to the clinic as soon as possible for testing. Home urinary pH is increasingly being recommended by urologists for patients with uric acid and calcium phosphate stones, as well as for patients on medication therapy for hypocitraturia. The study conducted by Desai and Assimos found that Chemstrip and litmus paper are more accurate than Multistix in determining urinary pH.19 When recommending a suitable dipstick for a patient, pharmacists need to keep in mind that the litmus paper is significantly cheaper than the Chemstrip and is an adequate test for patients who are attempting to maintain therapeutic urinary pH levels.
Handheld urinary pH monitors are becoming available on the market, but their accuracy and cost-effectiveness have yet to be tested. These monitors will probably prove useful in busy clinical settings or for patients who need to monitor urinary pH for the long-term.
Hematuria: Hematuria is generally defined as the presence of three or more red blood cells per high-power field (HPF).6 Hematuria causes pink or red urine and can be due to a number of factors ranging from strenuous exercise and UTIs to more severe conditions such as kidney or bladder cancers. Rarely, it is due to an inherited disorder known as porphyria, in which heme precursors are excreted in the urine. Exposure of the urine to sunlight in patients with porphyria will result in dark reddish urine that may slowly change to a brown color.
Hematuria is detected by the peroxidase activity of erythrocytes; however, hemoglobin and myoglobin can also catalyze this reaction, giving false-positive results.6 Additionally, dipstick testing is highly sensitive and may pick up levels as low as one to two red blood cells per HPF.22 Further microscopic evaluation is recommended to confirm the findings of a dipstick test.
Urine Specific Gravity: USG is a measure of the amount of substances dissolved in the urine and is expressed in grams/mL. It indicates the kidneys’ ability to concentrate urine and the patient’s dehydration status, with normal values between 1.001 and 1.305. Decreased USG is observed in diabetes insipidus and overhydration. USG less than 1.02 for over 12 hours is indicative of generalized renal impairment, nephrogenic diabetes insipidus, and symptoms of inappropriate antidiuretic hormone. End-stage renal disease is characterized by specific gravity values between 1.007 and 1.010.
On the other hand, dehydration, contaminated urine, diarrhea, and heart failure will result in a higher reading for USG. Increased USG is also noted in patients who have been given low molecular weight dextrans or IV radiographic dyes for radiographic studies.
Protein: The glomerular capillary wall filters low molecular weight proteins that are then reabsorbed and metabolized by the proximal tubule cells. The normal value for protein levels in the urine varies from 0 to 30 mg/dL; the proteins consist of albumin, serum globulins, and proteins secreted by the nephron. Sustained protein-uria from 10 to 20 mg/dL may be a symptom of renal damage and requires further investigation. Most dip-stick tests detect protein by production if color with bromphenol blue is sensitive to albumin, but these tests may not detect low levels of gamma-globulin and Bence-Jones protein. If a positive result is obtained for proteins in the urine, further investigation should be performed to confirm the diagnosis.23 Other causes of proteinuria include excessive exercise, exposure to cold, postural changes, pregnancy, febrile illness, UTIs, and diabetes mellitus.
Options for a differential diagnosis include a 24-hour urinary protein excretion (TABLE 5), spot urinary protein-creatinine ratio, precipitation tests, and microscopy. It has been shown that ranitidine (Zantac) can cause a false-positive test for proteinuria with Multistix. The manufacturers of Zantac therefore recommend testing proteinuria with sulfosalicylic acid. This is a more sensitive precipitation test that can detect albumin, globulins, and Bence-Jones protein at low concentrations.
| Extent of Renal Damage as Indicated by Amount of Protein in Urine | |||
| Protein Reading | Extent of Renal Damage | ||
| <1.0 g/24 h 1.0-3.5 g/24 h >3.5 g/24 h | Mild Moderate Severe nephrosis | ||
Glucose: Urine testing for glucose is useful for patients who find blood glucose monitoring difficult. Glucose levels should read below 130 mg/24 hours; higher values are often observed in diabetes mellitus, pregnancy, sepsis, tubular damage, Cushing’s syndrome, and liver and pancreatic disease. Most dipsticks employ the glucose oxidase reaction, are specific to glucose, and do not detect reducing sugars. To overcome this, patients may be tested further using the Clinitest that is based on Benedict’s copper reduction test.
Ketones: Ketone bodies are the breakdown product of fat, and their presence in urine (positive dipstick test) may be seen in patients with poorly managed diabetes mellitus, in anorexic patients, or in those who are dieting (e.g., with the Atkins diet). Dipstick tests or test tablets detect acetic acid through a reaction with sodium nitroprusside.
Urobilinogen and Bilirubin: Urobilinogen and bilirubin are only detected in the urine in liver dysfunction and indicate further evaluation. The Chemstrip-9 test uses diazonium dyes that turn red with urobilinogen and bilirubin. Kupka et al demonstrated that urine bilirubin and urobilinogen assays may offer a cost-effective way of predicting liver function abnormalities.24
Pregnancy Testing: Initial pregnancy tests can be performed on urine samples and later confirmed with a blood test. The levels of human chorionic gonadotropin (hCG) hormone double almost every 2.2 days during the first trimester of pregnancy, and these levels can be detected in the patient’s urine. Detectable levels start at 5 mIU/mL during the first week of gestation and rise to 100,000 mIU/mL at 2 to 3 months.2
Pregnancy test kits use monoclonal and polyclonal anti-hCG antibodies placed sequentially to detect the presence of hCG via a color change. The test area of the test strip is held under a stream of urine and then allowed to sit for about 1 minute. The specimen migrates from the test area by capillary action to a pad containing monoclonal anti-hCG antibody. If hCG is present, it forms a complex with the antibody, which then moves further to the test line. The test line is spotted with polyclonal hCG antibody, which binds the complex and causes a color change if hCG is detected. Some kits come with a control test line that will change color whether or not hCG is detected; this helps the patients ensure that they have performed the test correctly.
Sometimes, the levels of hCG are too low to be detected and patients may get a false-negative result. It is therefore important to advise the patient that she may need to perform a second test if she is testing very early on in the pregnancy. Some manufacturers provide two kits in the package so that patients may repeat the test a couple of weeks later if needed. Additionally, patients should test the first-morning midstream urine, when the levels of hCG are the highest. Patients taking diuretics and promethazine may also obtain a false-negative, whereas patients on anticonvulsants, anti-Parkinson drugs, hypnotics, and tranquilizers may get a false-positive result as well. Other causes of false-positive results are very rare but may indicate trophoblastic disease.
MICROSCOPIC ANALYSIS
Microscopic analysis reveals cells, pathogens, and contaminants visible under a HPF, and casts that can be viewed under a low-power field (LPF). The reference range for leukocytes, erythrocytes, and epithelial cells is 0 to 4/HPF. Normally urine does not contain any erythrocytes, as they are too large to pass through the renal tubules. Any red blood cells in the urine indicate hematuria and are a sign of a potential abnormality. White blood cells and transitional epithelial cells are visible in normal urine and only signal a problem if their concentrations increase. On the other hand, renal tubular cells are present in very low amounts and have normally disintegrated by the time the urine is voided. Intact renal tubular cells are an indication of renal tubular disease.
The presence of bacteria and yeasts in normal urine should be further analyzed using a culture. They may be a normal part of the microbial flora (e.g., bacteria), contaminants (e.g., yeast), or a sign of an infection.
Hyaline casts are formed from mucoprotein in the distal convoluted tubule and the collecting duct in the absence of cells. High numbers (>1/LPF) may suggest proteinuria, but such casts are normally present in lower numbers in the urine of most patients. Cellular casts contain leukocytes, erythrocytes or renal tubular cells, and sticky protein, and their presence is a symptom of renal inflammation, hematuria, or severe renal disease, respectively. When renal tubular cellular casts remain in the nephron for some time before being flushed out, they become granular casts, and then finally waxy casts. The granular and waxy casts are an indication of increasing renal damage.
The urine of healthy patients will commonly contain crystals of calcium oxalate and triple phosphate; their concentration is dependent on the urinary pH. However, the presence of cystine, tyrosine, and leucine crystals is abnormal and needs to be investigated.
In many cases, analyzing a urine sample microscopically is fairly challenging, and it may be difficult to identify and count the components even with a trained eye. This said, microscopic analysis is an indispensable part of urinalysis that can confirm a number of diagnoses made with dipstick testing.
CONCLUSION
Pharmacists play a vital role in guiding patients through the vast amount of information available today. Since urinalysis is an essential tool in clinical testing, it is important for modern pharmacists to have a working knowledge of these analytical procedures. Opportunities for counseling exist during the recommendation for OTC tests, interpretation of test results, referral of patients for further investigation, and adjustment of patients’ drug therapy.
