Urine Information and Courses from MediaLab, Inc.

A 72- year old woman had a history of recurrent urinary tract infections over the past several months, for which she had received different regimens of antibiotics including ampicillin, trimethoprim-sulfasoxazole, and ciprofloxacin.Relapses often occurred 10 days to two weeks after cessation of therapy.The current flare up, manifest by dysuria, lower abdominal pain and cloudy urine was accompanied by shaking chills and spiking fever.A sterile mid-stream urine specimen was sent to the laboratory for culture.

A chemical urinaylsis reagent strip, also called a dipstick, for screening urine is a narrow band of paper which has been saturated with chemical indicators for specific substances or properties. Depending on the product being used, chemical urinalysis reagent strips may include test indicators for glucose, bilirubin, ketones, specific gravity, blood, pH, protein, urobilinogen, nitrite, and leukocyte esterase. The results obtained from urine screening using chemical urinalysis strips can indicate the patient's carbohydrate metabolism status, kidney and liver function, urinary tract infection, and acid-base balance. Most chemical urinalysis reagent strips can be read visually and do not require instrumentation for automatic reading, though many laboratories utilize instruments for this purpose. When performing chemical urinalysis reagent strip analysis, the directions must be performed exactly. Accurate timing is paramount in order to achieve appropriate and optimal results. In addition, the reagent strips must be stored properly in their containers with the lid tightly closed to maintain reagent reactivity. It is always essential to utilize well-mixed urine which has been collected within 2 hours of analysis.Always read the package insert for your particular brand of chemical urinalysis reagent strip, as each manufacturer may have slightly different instructions and interpretations.

It is important for a quality urine sample to be sent to the laboratory in order for an accurate set of results to be produced. There are various types of urine samples that may be acceptable for urinalysis in the laboratory. The most common urine collection types are:Random Specimen: This is the specimen type which is sent to the laboratory for analysis most commonly. This type of urine sample is easy to obtain and is also readily available. As the name implies, the random specimen can be collected at any time. Patients should be careful not to touch the inside of the cup or cup lid to avoid any contamination.First Morning Specimen: This type of urine specimen is collected when the patient first wakes up in the morning. This is also occasionally called an 8-hour urine specimen.Midstream Clean Catch Specimen: This type of urine specimen concentrates on the reduction of contaminants in the urine sample by requiring special cleansing protocols. The urine midstream is then collected into a clean container.Other, less common types of urine specimens include:Timed Collection SpecimenCatheter Collection SpecimenSuprapubic Aspiration SpecimenPediatric SpecimenThe general procedure for using a reagent strip is outlined in this exercise. Each test on the strip will be discussed in detail in the remaining exercises.

Chemical reagent strips for urine measure pH using methyl red and bromthymol blue indicator dyes. The color change that occurs in this test area correlates with the urine's pH. Sensitivity to pH ranges from 5.0 (acid pH) to 9.0 (alkaline pH) on a typical urine reagent strip. Physiological urine pH ranges from 5.0 to 8.0. With an increase in urinary pH, the indicators bromothymol blue and methyl red, changes from orange to green and blue.

Testing for protein in the urine is based on the phenomenon called the "Protein Error of Indicators" (ability of protein to alter the color of some acid-base indicators without altering the pH). In a solution void of protein, tetrabromphenol blue, buffered at a pH of 3, is yellow. However, in the presence of protein, particularly albumin, the color changes to green, then blue, depending upon the concentration. This method is more sensitive to albumin than to globulin, detecting as little as 5 mg albumin/dL urine. Bence Jones protein and mucoprotein are examples of globulin components that are sometimes present in urine, but are not distinguishable by the chemical reagent strip method for urine protein. False positive results can occur when testing for urine protein. A urine specimen that has remained at room temperature for an extended period of time may produce a false-positive protein result on a reagent strip. A false positive may also occur in the presence of bacterial contamination, alkaline medication, quaternary ammonium compounds such as disinfectants or drugs, and with skin cleansers containing chlorhexidine.

Semiquantitative tests are used in some laboratories to confirm the presence of protein in a specimen when the result is positive on the urine chemical reagent strip. Tests that are used for confirmation include: sulfosalicylic acid (SSA), heat and acetic acid, nitric acid ring test, and Roberts' Ring Test. Any one of these procedures may be used for confirmation of the presence of protein. NOTE: A protein result on a urine chemical reagent strip that is greater than a trace may be an indication of proteinuria.

Individuals with diabetes mellitus may excrete small amounts of albumin in the urine (microalbumin) which may signal the beginning of reduced glomerular filtration. Stabilizing the blood glucose level at this time may delay progression of diabetic nephropathy. Both type I and type II diabetes mellitus are leading causes of renal failure. Microvascular damage caused by excessive renal exposure to glucose can lead to diabetic nephropathy. By the time the urine protein level reaches the 30 mg/dL level that is necessary for detection by routine reagent strips, damage to the kidneys may have already occurred. Reagent strips are available that use a dye-binding technique rather than the traditional protein-error of indicators principle. These strips are more sensitive and specific for albumin, detecting levels as low as 8 mg/dL.Women in the last month of pregnancy may develop proteinuria as the first sign of impending eclampsia. Eclampsia is the gravest form of toxemia of pregnancy. The presence of protein in this situation must be evaluated by the physician in conjunction with other clinical symptoms.Benign transient proteinuria may be the result of: exposure to cold, strenuous exercise, dehydration, and/or high fever. Benign transient proteinuria may also occur during the acute phase of a severe illness. Patients over the age of 60 have a greater chance of having protein in their urine. Occult malignancies and glomerulonephritis, that occur more frequently in the elderly, may be signaled by the presence of proteinuria. Orthostatic proteinuria is a condition seen most often in young adults. The condition may be caused by pressure on the renal nerve. When this condition is suspected, two urine specimens are tested. One specimen is collected upon arising in the morning, and the second is collected several hours later. When this condition is present, the first morning specimen, after the patient has been in a supine position, will be negative for protein. The second specimen, taken after the patient has been upright for several hours, would be positive for protein.

False Positives:False positive results can be attributed to strong oxidizing agents such as hydrogen peroxide (H2O2) or bleach (hypochlorite). False Negatives:False negative results occur when elements present in the urine interfere with either the enzymatic reaction or prevent the oxidation of potassium iodide. Examples of some substances that may produce false negative results, depending on the reagent strip that is used, include: large quantities of ketonesaspirinascorbic acid > 50 mg/dL levadopa5-hydroxyindoleacetic acidhomogentisic acidsodium fluoride ( a preservative) A specific gravity higher than 1.020 may lower glucose reagent sensitivity, especially in the presence of a high urine pH. Exposing reagent strips to excess humidity may also reduce glucose reagent reactivity.NOTE: Check the package insert of the reagent strips used in your laboratory for interfering substances that may affect glucose results.

In a healthy individual, almost all of the glucose filtered by the renal glomerulus is reabsorbed in the proximal convoluted tubule. The amount of glucose reabsorbed by the proximal tubule is determined by the body's need to maintain a sufficient level of glucose in the blood. If the concentration of blood glucose becomes too high (160-180 mg/dL), the tubules no longer reabsorb glucose, allowing it to pass through into the urine. It is important to note that glucose may appear in the urine of healthy individuals after consuming a meal that is high in glucose. Fasting prior to providing a sample for screening eliminates this problem. Conditions in which glucose levels in the urine are above 100 mg/dL and detectable include: diabetes mellitus and other endocrine disordersimpaired tubular reabsorption due to advanced kidney diseasepregnancy - glycosuria developing in the 3rd trimester may be due to latent diabetes mellituscentral nervous system damagepancreatic diseasedisturbances of metabolism such as, burns, infection or fractures

When the body breaks down fat for energy, three intermediate products are formed. These products, collectively referred to as ketones, include acetone, acetoacetic acid, and beta-hydroxybutyric acid. Normally, the body gets the energy it needs from carbohydrates in the diet. However, stored fat is broken down and ketones are produced and appear in the urine if the diet does not contain enough carbohydrate to supply the body with glucose for energy or if the body cannot use glucose properly.

Ketone bodies are usually absent in urine, but low levels may be detected during conditions of physiological stress such as fasting, rapid weight loss, frequent strenuous exercise or prolonged vomiting. The presence of ketones in these situations is due to either inadequate intake or increased loss of carbohydrates. High levels of ketones are present in the urine of individuals with uncontrolled diabetes. In diabetes the ketones are present because the body's ability to metabolize carbohydrates is defective.

Ketone bodies are usually absent in urine. The presence of ketones in the urine most likely indicates that the body is using fats rather than carbohydrates for energy. For example, high levels of ketones may be present in the urine of individuals with uncontrolled diabetes because the body's ability to metabolize carbohydrates is defective. Detecting the presence of ketones in the urine is a valuable aid to managing and monitoring individuals with diabetes mellitus. Ketonuria is an indication that the insulin dose needs to be increased. Electrolyte imbalance and dehydration may occur when ketones accumulate in the blood. If these conditions are not corrected by adjusting the dose of insulin, the patient may develop ketoacidosis and ultimately diabetic coma. Low levels of ketones may also be detected in the urine during conditions of physiological stress such as fasting, rapid weight loss, frequent strenuous exercise or prolonged vomiting. The presence of ketones in these situations is due to either inadequate intake of carbohydrates or increased loss of carbohydrates.

Bilirubin, a product of hemoglobin catabolism, is characterized by its distinctive yellow pigment. The presence of bilirubin in urine is always abnormal. In most healthy individuals the amount of conjugated bilirubin excreted is not detected by the strips. In cases when bilirubin is elevated and is conjugated, it will be detected by the test strip.It is important to note that unconjugated bilirubin cannot be excreted by the kidneys because it is bound to albumin and is not soluble in water. In the liver, bilirubin combines with glucuronic acid through the action of a glucuronyl transferase to form water soluble bilirubin diglucuronide. Under normal circumstances, conjugated bilirubin passes from the bile duct and then to the intestinal tract. Intestinal bacteria reduce conjugated bilirubin to urobilinogen. Approximately half of the urobilinogen is excreted in the feces; most of the other half is recirculated through the liver. A small amount of urobilinogen bypasses the liver and is excreted in the urine.

False Positive BilirubinFalse positive results may occur when patients are on large doses of chloropromazine, and may occur in the presence of metabolites of phenazopyridine. When these compounds are present, the urine becomes red. Metabolites of Lodine® (etodolac) may cause false positive or atypical results. False Negative BilirubinFalse negative bilirubin dipstick results are often due to testing a specimen that is not fresh. Bilirubin breaks down when exposed to light. Indoxyl sulfate (Indican) can produce a yellow orange-to-red color response which may interfere with the interpretation of a positive or negative reaction. Positive nitrites due to a urinary tract infection may also cause a false negative result.

Liver damage or an obstructed bile duct allows conjugated bilirubin to enter the circulation and ultimately to appear in the urine. Patients with clinical jaundice due to hepatitis or cirrhosis will have bilirubinuria. If the jaundice is due to red cell destruction, there is an increase in unconjugated bilirubin which the kidneys cannot excrete.

The test for blood on the urine chemical reagent strip is based on the peroxidase-like activity of hemoglobin which catalyzes the reaction of cumene hydroperoxide and 3, 3', 5, 5' tetramethylbenzidine. The test is sensitive to free hemoglobin, myoglobin and a minimum of 5 intact red cells per microliter of urine.

Blood is normally not present in the urine of healthy individuals, apart from blood during menses that may be detected in urine samples from females, Hematuria is associated with renal or genital disorders in which the bleeding is the result of irritation to the involved organs or some type of trauma. Examples include:Renal calculiPyelonephritisGlomerulonephritisTumorsTraumaExposure to toxic chemicals or drugsStrenuous exerciseHemoglobinuria may be due to the lysis of red blood cells within the urinary tract. This can be caused by intravascular hemolysis, as the hemoglobin is filtered through the glomeruli. In a healthy, normal individual, the hemoglobin molecule attaches to haptoglobin and bypasses the kidney filtration system. When the hemoglobin/haptoglobin system is overwhelmed, hemoglobin passes into the urine. Hemoglobinuria may be associated with:Hemolytic anemiaSevere burnsTransfusion reactionInfection Strenuous exercise

The nitrites portion of the chemical reagent strip provides a rapid screening test for the presence of gram-negative bacteria that are often responsible for urinary tract infections. Urine cultures are still needed to confirm the diagnosis and monitor any urinary tract or kidney infection. Diagnosis and treatment of cystitis (bladder infection) is important because, if left untreated, it may result in kidney damage, impairment of renal function, hypertension and/or septicemia.

This test is sensitive to 0.06-0.1 mg/dL nitrite ion in urines with a low specific gravity and with ascorbic acid concentrations of less than 25 mg/dL. Pink spots or pink edges should not be interpreted as a positive result because some medications can color urine red or turn red in an acid environment. Any degree of uniform pink color should be considered positive, suggesting the presence of 105 organisms/mL. Detection of low levels of nitrite ion may be enhanced by comparing the activated test strip to a white background. It is important to note that color development is NOT proportional to the number of bacteria present. The test is specific for nitrites and does not react with any other substances normally present in urine. Negative results do not necessarily rule out a urinary tract infection with yeasts or gram-positive bacteria unable to reduce nitrites.

Early detection of bacteria is important in order to prevent cystitis from developing into inflammation or infection involving the kidney and renal pelvis. The nitrite portion of the test strip can be used to screen individuals who are at risk for developing urinary tract infections, such as diabetics, persons with recurrent infections, or pregnant women. The test is also useful in evaluating the success of antibiotic therapy that is used to treat a bladder infection.

The test for urobilnogen is based on the Ehrlich Aldehyde Reaction. P-dimethylaminobenzaldehyde in an acid medium with a color enhancer reacts with urobilinogen to form a pink-red color. The urine chemical reagent strip reactivity increases with increasing temperature. The optimum temperature for testing is 22° - 26°C. A freshly voided sample is always best for ensuring optimal results.

Urinary urobilinogen may be increased in the presence of a hemolytic process such as hemolytic anemia. It may also be increased with infectious hepatitis, or with cirrhosis. Comparing the urinary bilirubin result with the urobilinogen result may assist in distinguishing between red cell hemolysis, hepatic disease, and biliary obstruction, as shown in the table below:ConditionUrine Bilirubin ResultUrine Urobilinogen ResultHemolytic diseaseNegativeIncreasedHepatitic diseasePositive or negativeIncreasedBiliary obstructionPositiveNormal* *Urine chemical reagent strip methods cannot distinguish normal urobilinogen from absent urobilinogen, as might be seen in complete biliary obstruction.

If leukocyte esterase is detected on a urine chemical reagent strip, a color change occurs on the reagent pad after the strip is dipped in the urine sample. Be sure to follow the manufacturer's directions for read-time and test interpretation. A positive leukocyte esterase test indicates the presence of granulocytic white blood cells. Lymphocytes do not contain granules, and would not produce a positive leukocyte esterase test. Positive results should be confirmed by performing a microscopic examination on the sediment; keeping in mind that white blood cells may be absent if they are lysed, yet have released their esterases into the urine specimen. Positive results may occasionally be found in random specimens from females due to contamination of the specimen by vaginal discharge.

Using the esterase test in conjunction with pH, protein and nitrite provides a combination of tests which can screen for the presence of bacterial infections in the urinary system.

The chemical reagent strip measures specific gravity (SG) in increments of 0.005 with readings from 1.000 to 1.035. The test principle is based on a change in pKa (the negative log of the acid disassociation) of certain pretreated electrolytes (methylvinyl ether/maleic anhydride) in relation to ionic concentration of the urine. These electrolytes in the reagent area contain acid groups which disassociate according to the ionic concentration of the specimen. The more ions in the specimen, the more acid groups will become disassociated, releasing hydrogen ions and causing a more acid pH. The reagent area contains a pH indicator (bromthymol blue) which demonstrates the change in pH. The higher the specific gravity of the urine specimen, the more acidic the reagent area will become. The colors of the reagent area will range from deep blue-green in urines of low ionic concentration to green-to-yellow green in urines of increasing ionic concentration, and consequently, higher specific gravity.

Measurement of specific gravity provides information regarding a patient's state of hydration or dehydration. It also can be used to determine loss of renal tubular concentrating ability. There are no "abnormal" specific gravity values. This test simply indicates how concentrated the urine is.

The reagent strips must be handled and stored properly in order to ensure that results are accurate. The following precautions should be observed:Store strips according to the manufacturer's recommendation. DO NOT expose strips to moisture, volatile fumes, or direct sunlight (emphasized in the image on the right). Remove only enough strips for immediate use and immediately recap the bottle. Avoid contamination of test strips. Do not touch the test areas with fingers and do not lay the test strips directly on the workbench. DO NOT use discolored strips. Compare the color of the unused strip to the negative area on the color chart provided by the company. The color should be similar. Check the expiration date. Re-label the container with a revised expiration date, if the manufacturer states a shortened usage period once the container has been opened. Reagent strips must be tested periodically (frequency defined by the laboratory) for clinical reactivity with normal and abnormal urine controls. Urine controls are available commercially or may be prepared and preserved in-house.

Use a fresh, well-mixed, uncentrifuged urine. Hold the reagent strip by the opposite end from the test areas and dip the stick into the specimen so that all test areas are immersed in the specimen. Remove the stick immediately. Prolonged immersion in the sample may wash out the test reagents. Hold strip in a horizontal position and run the edge of the strip against the rim of the urine container or touch the long edge of the strip to absorbent toweling to remove excess urine (do not blot the strip). Maintain the strip in a horizontal position to prevent mixing of reagent chemicals. If you are using a dipstick reader, place the strip immediately onto the tray of the reader.Replace the cap on the container to prevent deterioration of remaining strips If you are reading the tests manually, proceed with these instructions:Observe the reagent pads at the specified time periods. Color changes that occur after the stated maximum read time are not valid. Hold the strip close to the chart and compare the colors to read the results. A good light source facilitates accurate reading.

Dipsticks measure pH using methyl red and bromthymol blue indicator dyes. The color change that occurs in this test area correlates with the urine's pH. Sensitivity to pH ranges from 5.0 (acid pH) to 9.0 (alkaline pH) on a urine reagent strip. Physiological urine pH ranges from 5.0 to 8.0.

Urine pH results must be evaluated in conjunction with a patient's medical condition and clinical history. Factors to be considered include:Respiratory and metabolic statusRenal function Crystal or calculi formationDietThe table below summarizes dietary, medical, and artifactual conditions that may affect urine pH:ConditionAcid pHAlkaline pHHigh meat dietXVegetarian dietXRespiratory/metabolic acidosisXRespiratory/metabolic alkalosisXHypochloridemiaXHigh concentration of urine glucoseXBacterial infection caused by urease-producing bacteriaXProlonged storage of specimen at room temperature, allowing multiplication of urease-producing bacteriaX (above 8.0)Improper procedural technique; excess urine left on reagent strip, allowing acid buffer in protein pad to run over into adjacent pH pad (refers to some reagent strip configurations)X

A urine specimen that has remained at room temperature for an extended period of time may produce a false-positive protein result on a reagent strip. A false positive may also occur in the presence of bacterial contamination, alkaline medication, or quaternary ammonium compounds such as disinfectants or drugs, and with skin cleansers containing chlorhexidine.

Semiquantitative tests are used in some laboratories to confirm the presence of protein in the specimen when the result is positive on the urine dipstick. Tests that are used for confirmation include: sulfosalicylic acid (SSA); heat and acetic acid; nitric acid ring test; and Roberts' Ring Test. Any one of these procedures may be used for confirmation of the presence of protein. A protein dipstick result that is greater than a trace may be an indication of proteinuria.

Proteinuria related to kidney impairment may be due to glomerular membrane damage caused by toxic agents, immune complexes found in lupus erythematosus, or streptococcal glomerulonephritis. The amount of protein present in urine samples from patients with glomerular damage usually ranges from 10-40 mg/dl. If the urinary protein is due to a disorder that affects tubular reabsorption, the urine protein quantities will be much greater. In patients with multiple myeloma, proteinuria is due to the excretion of the Bence Jones protein. This low molecular weight protein produced by a malignant clone of plasma cells circulates in the blood and is filtered in the kidneys in quantities exceeding the tubular capacity. This excess protein is excreted in the urine.

Patients over the age of 60 have a greater chance of having protein in their urine. Occult malignancies and glomerulonephritis, that occur more frequently in the elderly, may be signaled by the presence of proteinuria. Orthostatic proteinuria is a condition seen most often in young adults. The condition may be caused by pressure on the renal nerve. When this condition is suspected, two urine specimens are tested. One specimen is collected upon arising in the morning, and the second is collected several hours later. When this condition is present, the first morning specimen, after the patient has been in a supine position, will be negative for protein. The second specimen, taken after the patient has been upright for several hours, would be positive for protein.

False negative results occur when elements present in the urine interfere with either the enzymatic reaction or prevent the oxidation of potassium iodide. Examples of such substances include: large quantities of ketones aspirin ascorbic acid > 50 mg/dL with some reagent strips levadopa 5-hydroxyindoleacetic acid homogentisic acid sodium fluoride ( a preservative)A specific gravity higher than 1.020 may lower glucose reagent sensitivity, especially in the presence of a high urine pH. Exposing reagent strips to excess humidity may also reduce glucose reagent reactivity.Check the package insert of the reagent strips used in your laboratory for interfering substances that may affect glucose results.

In the healthy individual, almost all of the glucose filtered by the renal glomerulus is reabsorbed in the proximal convoluted tubule. The amount of glucose reabsorbed by the proximal tubule is determined by the body's need to maintain a sufficient level of glucose in the blood. If the concentration of blood glucose becomes too high (160-180 mg/dL), the tubules no longer reabsorb glucose, allowing it to pass through into the urine. It is important to note that glucose may appear in the urine of healthy individuals after consuming a meal that is high in glucose. Fasting prior to providing a sample for screening eliminates this problem.

When the body breaks down fat for energy, three intermediate products are formed. These products, collectively referred to as ketones, are acetone, acetoacetic acid, and beta-hydroxybutyric acid. Normally, the body gets the energy it needs from carbohydrates in the diet. However, stored fat is broken down and ketones are produced and appear in the urine if the diet does not contain enough carbohydrate to supply the body with glucose for energy or if the body cannot use glucose properly.

Ketone bodies are usually absent in urine, but low levels may be detected during conditions of physiological stress such as fasting, rapid weight loss, frequent strenuous exercise or prolonged vomiting. The presence of ketones in these situations is due to either inadequate intake or increased loss of carbohydrates. High levels of ketones are present in the urine of individuals with uncontrolled diabetes. In diabetes the ketones are present because the body's ability to metabolize carbohydrates is defective.

The presence of excess moisture/humidity can cause the ketone reagent to become nonreactive, resulting in a false negative test for ketones. Urine specimens should not remain at room temperature if testing is delayed because ketones are unstable at room temperature.

Bilirubin, a product of hemoglobin breakdown, is characterized by its yellow pigment. The presence of bilirubin in urine is always abnormal. It is important to note that unconjugated bilirubin cannot be excreted by the kidneys because it is bound to albumin and is not soluble in water. In the liver, bilirubin combines with glucuronic acid through the action of a glucuronyl transferase to form water soluble bilirubin diglucuronide. Under normal circumstances, conjugated bilirubin passes from the bile duct and then to the intestinal tract. Intestinal bacteria reduce conjugated bilirubin to urobilinogen. Approximately half of the urobilinogen is excreted in the feces; most of the other half is recirculated through the liver. A small amount of urobilinogen bypasses the liver and is excreted in the urine.

False positive results may occur when patients are on large doses of chloropromazine, and may occur in the presence of metabolites of phenazopyridine. When these compounds are present, the urine becomes red. Metabolites of Lodine® (etodolac) may cause false positive or atypical results.

Liver damage or an obstructed bile duct allows conjugated bilirubin to enter the circulation and ultimately to appear in the urine. Patients with clinical jaundice due to hepatitis or cirrhosis will have bilirubinuria. If the jaundice is due to red cell destruction, there is an increase in unconjugated bilirubin which the kidneys cannot excrete.

The test for blood on the urine reagent strip is based on the peroxidase-like activity of hemoglobin which catalyzes the reaction of cumene hydroperoxide and 3, 3', 5, 5' tetramethylbenzidine. The test is sensitive to free hemoglobin, myoglobin and a minimum of 5 intact red cells per microliter of urine.

False negative results may occur with some methods when the concentration of ascorbic acid is greater than 5 mg/dL. The sensitivity of the blood portion of the test strip is decreased in specimens with a high specific gravity and increased protein. High levels of nitrites may delay the reaction, causing a false negative to be reported. If the pH of a urine sample is below 5, hemolysis of red cells as part of the test reaction is inhibited which results in a false negative reaction. An improperly mixed specimen may test negative if the red blood cells are in the sediment.

Bladder infections are usually caused by gram-negative bacteria. These bacteria reduce nitrates derived from food to nitrites when urine remains in the bladder three to four hours, sufficient time for this reaction to occur. Nitrite is not present in urine under normal circumstances. When present, nitrites react with p-arsanilic acid to produce a diazonium compound. The diazonium compound in turn couples with 3-hydroxy-1,2,3,4 tetrahydrobenzo-(h)-quinolin to produce a pink color. A first morning, clean, voided midstream specimen is optimal for detecting nitrites in urine.

A false positive nitrite test result may occur when a urine specimen has remained at room temperature for an extended period of time, allowing bacterial contaminants to multiply and produce measurable levels of nitrites. Interference from some medications that cause the urine to become red or orange may lead to an incorrect reading of positive for nitrite.

Urobilinogen is a byproduct of hemoglobin breakdown. It is produced in the intestinal tract as a result of the action of bacteria on bilirubin. Almost half of the urobilinogen produced recirculates through the liver and then returns to the intestines through the bile duct. Urobilinogen is then excreted in the feces where it is converted to urobilin. As the urobilinogen circulates in the blood to the liver, a portion of it is diverted to the kidneys and appears as urinary urobilinogen. Up to 1 mg/dL or Ehrlich unit of urobilinogen is present in normal urine. A result of 2.0 mg/dL represents the transition from normal to abnormal and the patient should be evaluated further. It is important to note that the reagent strip cannot determine the absence of urobilinogen.

Due to the instability of urobilinogen, a false negative result may occur using a dipstick method if the urine specimen has remained at room temperature for an extended period of time in the light. A false negative result may also occur if formalin is present.

Granulocytic white blood cells in a urine sample suggest the presence of a urinary tract infection. Granulocytes, which include neutrophils, basophils and eosinophils, contain esterases. These esterases catalyze the strip reagent indoxylcarbonic acid ester to release indoxyl. Indoxyl reacts with a diazonium salt to produce a purple color. The intensity of the color produced is proportional to the amount of enzyme present.

A false positive result may occur in the presence of strong oxidizing agents in the collection container. In random urine specimens from women, a positive result for leukocyte esterase may be due to a source external to the urinary tract. Other urine sediment findings such as bacteria, squamous or renal epithelial cells, lymphocytes or red blood cells do not contain esterases, and would not produce a positive leukocyte esterase test.

The reagent strip measures specific gravity (SG) in increments of 0.005 with readings from 1.000 to 1.035. The test principle is based on a change in pKa (the negative log of the acid disassociation) of certain pretreated electrolytes (methylvinyl ether/maleic anhydride) in relation to ionic concentration of the urine. These electrolytes in the reagent area contain acid groups which disassociate according to the ionic concentration of the specimen. The more ions in the specimen, the more acid groups will become disassociated, releasing hydrogen ions and causing a more acid pH. The reagent area contains a pH indicator (bromthymol blue) which demonstrates the change in pH. The higher the SG of the urine specimen, the more acidic the reagent area will become. The colors of the reagent area will range from deep blue-green in urines of low ionic concentration to green-to-yellow green in urines of increasing ionic concentration, and consequently, higher SG.

The reagent strip protein method is based on the principle of "protein-error-of-indicators." It produces a visible colorimetric reaction that is capable of detecting most instances of proteinuria. Falsely elevated results can occur if the urine sample is visibly bloody.At one time, sulfosalicylic acid (SSA) was used to confirm all positive protein reagent strip results. However, this no longer routinely done. SSA is a precipitation method that reacts with all forms of protein. False-positives can occur. Any substance that is precipitated by acid will produce false-positive SSA results, including radiographic dyes, cephalosporins, penicillins, and sulfonamides. SSA may be used as a secondary protein detection method if the urine is highly colored so that the colorimetric reaction is masked on the reagent strip.

The presence of low levels of albumin (microalbumin) in the urine is an important finding in an individual with either type 1 or type 2 diabetes. The development of clinical nephropathy leads to reduced glomerular filtration and eventually may lead to renal failure. For this reason, early detection of microalbumin is important in order to avert renal complications in a diabetic patient. The presence of microalbuminuria has also been associated with an increased risk for cardiovascular disease. Reagent strips that are used for routine urinalysis cannot detect low levels of albumin excretion (1 to 2 mg/dL). Special reagent strips that are sensitive for these low levels of albumin are useful for periodic monitoring of patients with diabetes, hypertension, or peripheral vascular disease.

Measure the amount of urine that is specified in the test procedure onto the center of the test mat Place one reagent tablet onto the center of the mat Place one drop of water onto the mat, wait 5 seconds and add a second drop so that the water run off the tablet onto the mat.

Bilirubin may be present in the urine when bile duct obstruction, liver disease, or liver damage is present. Bilirubinuria can be detected before other clinical symptoms, such as jaundice, are present or recognizable. The detection of small quantities of bilirubin is very important in early diagnosis of obstructive and hepatic jaundice. The urine bilirubin test is also useful in the differential diagnosis of obstructive jaundice (positive for bilirubinuria) vs. hemolytic jaundice (negative for bilirubinuria).

Since detectable amounts of bilirubin are not normally present in urine, results of the Ictotest® are reported as "positive" or "negative", there is no quantitation. The sensitivity of Ictotest® is better than dipstick methods or the Harrison test. Ictotest® will detect as little as 0.05-0.10 mg bilirubin/dl urine, making it the procedure of choice for confirming bilirubin in urine specimens.

Urine reagent strips are normally adequate for urine screening, but occasionally, it may be necessary to perform a secondary procedure to ensure the accuracy of the test result. Confirmatory or secondary procedures are usually performed for one or more of these reasons: To confirm a result that has been obtained on the reagent strip. To obtain a result from a highly pigmented urine that masks the result on the reagent strip. To test for a specific analyte (or analytes) that are not included in the specificity of the reagent strip test. For example, the glucose reagent strip test is specific for glucose, but you want to test for other reducing substances.

Although glucose is the sugar most commonly tested for in urine, normal human urine can contain small amounts of galactose, lactose, fructose, xylose, and other pentoses. Galactosuria, an abnormal amount of galactose in the urine, occurs in infants with a congenital metabolic defect. Lactose may be found in the urine of nursing women and during late pregnancy. All of these sugars, including glucose, are reducing substances.

The Clinitest® method can detect reducing substances in the urine up to 2 g/dL. When the amount of sugar is over 2 g/dL (often expressed as 2%), a “pass through” phenomenon occurs. Pass through appears as rapid color changes through green, tan, and orange, and then a reversion in color back to the brownish color. This reversion in color indicates levels of reducing substances greater than 2 g/dL. Even a fleeting orange color should be recorded as “greater than or equal to 2 g/dL.” It is vital that you watch the boiling and color changes throughout the entire reaction so that a "pass through" is not missed.

Enzyme-based methods are most often used to detect/monitor urine glucose and copper reduction methods are used to detect all reducing substances. Most enzyme tests use the enzyme glucose oxidase. which is impregnated on a dipstick along with a chromagen. These enzyme-based dipstick tests are specific for glucose.

Ketonuria occurs when fatty acids are moved from triglyceride stores in the body in response to inadequate intake or availability of carbohydrates. Under conditions of abnormal carbohydrate metabolism, such as occurs in diabetes mellitus, ketones accumulate in the blood (ketonemia) and are excreted in the urine (ketonuria). The accumulation of ketones is often the cause of acidosis and coma in diabetics. Ketonuria is also associated with:StarvationDigestive disturbancesDietary imbalance (high fat/low carbohydrate diet)EclampsiaProlonged vomiting and diarrheaGlycogen storage diseasesSevere, sustained exerciseFeverProlonged exposure to cold temperaturesKetones are mildly toxic to the body, tending to interfere with the excretion of uric acid, produce mild depression of the central nervous system, and cause acidosis.

Screening for ketonuria is useful in following the effects of treatment for diabetes and in judging the severity of acidosis. Large amounts of ketones will appear in the urine before serum ketone levels are elevated.

Acetest® tablets can be used for the semiquantitation of ketones in urine, serum, or whole blood. However, an assay for serum/plasma beta-hydroxybutyrate (BHB) is very important in the assessment of diabetic ketoacidosis as BHB is the predominant ketone body and the most sensitive marker for detection of acidosis. Because the nitroprusside method (reagent strips and Acetest) do not measure BHB, a specific test for this ketone body is needed.Urine to be screened for ketone bodies using the Acetest method should be tested immediately or refrigerated in a closed container since acetone is lost to the air if the sample is held at room temperature for a prolonged period.

The last type of collection is the observed collection. In this type of collection both the donor and "observer" enter the collection stall. Observed collections afford less privacy in order to guard against the donor using items which are designed specifically to beat the testing process. Under the Department of Health and Human Services mandatory guidelines for Federal workplace drug testing the observer must directly watch the urine go from the donor's body into the collection container. The use of mirrors or video cameras is not permitted.The observed collection is expanded under the Department of Transportation's 49 CFR § 40. After entering the stall the observer requests the donor to: Raise shirt, blouse, dress / skirt as appropriate, above the waist, just above the navel, Lower clothing and underwear to mid-thigh, and, Then turn around to show the observer that the donor does not have a prosthetic device. After the observer has determined that the donor does not have a prosthetic device, the donor is permitted to return clothing to proper position. The observer must personally watch the urine go from the donor's body into the collection container. The use of mirrors or video cameras is not permitted.

The Non-Federally Regulated Custody and Control Form is most often used in clinics and hospital emergency rooms when drug abuse is suspected, or by companies participating in their state's drug-free workplace program. Be aware that some states mandate the use of a special CCF for their drug-free workplace program. There are significant differences between the Federally Regulated CCF and the Non-Regulated CCF. You are strongly encouraged to review the difference between the two. Unless there are extenuating (which we will discuss next), remember that the two forms are not interchangeable. The Federally Regulated CCF can be used only for urine collections required by the Department of Transportation drug testing program.

Regardless whether you are collecting a Federally Regulated or a Non-Federally Regulated urine drug screen, there are five areas which demand specific prerequisites or conditions prior to performing a proper collection. These are: Requirements for the collection site. Supplies needed to conduct a collection. Criteria that must be met by collectors. Complete and accurate documentation. Proper identification of the donor.We will explore each of these in more detail over the next several pages.

All collection sites must meet the following security requirements: Must be able to prevent unauthorized access to the site during collection. Ensure that the donor does not have access to items that could be used to adulterate or dilute the specimen (e.g. soap, water, cleaning agents, etc.) Secure faucets, toilet tank tops, and other appropriate areas with tamper-evident tape if necessary. Ensure that the donor is at all times under the supervision of the collector or other collection site personnel. Provide for the secure handling and storage of specimens. (Specimens should be stored at 4-6° C. The refrigerator used should not be readily accessible to the general public and should be used only for the storage of urine drug screens and other clinical specimens. The refrigerator should be marked with a biohazard sign. No food or drink should ever be placed in the refrigerator.)

One of the most important aspects of a urine drug screen collection is the correct identification of the donor. It is the responsibility of the donor to provide the collector appropriate identification upon arrival at the collection site.Acceptable forms of identification include: A photo identification such as a driver's license, an employee badge, or any other picture ID issued by either a federal, state, or local government agency. Identification made by an employer or a representative of the employer. In this latter case, the employer or employer representative can describe the donor to the suitability of the collector via a phone call.

After the donor has handed his or her specimen to the collector, the collector now opens the specimen bottle(s). Make sure that the security seals for the specimen bottle(s) are only opened in the presence of the donor.The collector, not the donor, disperses the urine specimen as follows:Federally Regulated (DOT):A minimum of 15 mL into one specimen container, a minimum of 30 mL into the second specimen container.Non-Regulated:A minimum of 30 mL into just one of specimen containers.

After a positive identification has been made, invite the donor into the area where the collection will be conducted. Be pleasant, but professional. Introduce yourself and generally explain the collection procedure. Be prepared to accommodate donors who do not speak English. Never argue with the donor or be judgmental. Always remember that you are a professional. Conduct yourself in that manner. Ask the donor to remove any unnecessary out clothing such as a coat, jacket, hat, etc., and to leave any briefcase, purse, or other personal belongings with the outer clothing. The donor may retain his or her wallet. If the donor asks for a receipt for any belongings left with the collector, the collector must provide one. Direct the donor to empty his or her pockets and display the items to ensure that no items are present that could be used to adulterate or dilute the specimen or be used as a substitute. If nothing is there, the donor may return the items to his or her pockets.

If a donor for pre-employment testing fails to appear, does not provide a urine specimen, or leaves the collection site before the collection process begins (e.g. before being given a collection cup), this is not considered a refusal to test.If a donor refuses to sign the donor certification on the pink copy of the CCF or to initial the security strips, this is not considered a refusal to test.If a donor refuses to provide an ID or Social Security Number, this is not considered a refusal to test.If a donor during the three (3) hour waiting period for a "shy bladder" refuses to drink any liquids, this is not considered a refusal to test.

If the donor provides an initial insufficient specimen, the collector discards the insufficient specimen and notes in the Remarks section of the CCF when the donor provided the insufficient specimen. If in an insufficient specimen there is enough urine to activate the temperature strip, and the specimen is out of temperature range, the collector will initiate the next collection under direct observation.The "shy bladder" procedure also goes into effect if from an earlier collection it was determined that an observed collection must be made and the donor is unable to provide a specimen.If the donor is unable after three hours to provide a specimen, the collector must discontinue the collection and note that no specimen was obtained in the "check" box and in the Remarks section of the CCF. Notation should also be made as to the quantity of specimen that was collected, if any, and amount of fluids the donor was given to drink. The collector must immediately notify the DER.

Scenario 5: The collector notices that the urine the donor just handed to her has a very strong smell like that of a cleaning product such as bleach. Collector's response: The collector completes the collection in the usual manner and prepares the specimen for shipment. The collector explains the situation to a supervisor. If the supervisor concurs that an observed specimen should be collected, the collector explains to the donor that because of the strong, unusual smell, the first specimen is suspect for adulteration and that a directly observed collection will be done. A new CCF is initiated. The collector marks on the CCF that the collection is observed and notes under Remarks why it is observed. The collector also notes the control number of the suspect collection. The observed specimen along with the suspect specimen are both shipped to the laboratory in separate plastic tamper-resistant bags. In addition to an unusual smell, other indications of adulteration might be an unusual color that cannot be explained by medication, particles or debris in the urine, and a heavy or thick foam that is inconsistent with urine.

You, as a collector, have a great deal of responsibility in the collection of urine for drug testing. It is imperative that you know, understand, and stay current with the rules and regulations. Do the very best you can to make every collection "error free." A collection site must be ready to demonstrate that it satisfies all requirements. Guidelines now mandate that "Federal agencies" inspect each year up to five percent of randomly selected sites used by the agency.

Ketoacidosis is always a serious complication for type 1 diabetics. Due to lack of uptake of glucose into cells by insulin, proteins and fats are utilized as energy sources. This results in excess acetyl CoA which is converted to ketone bodies. A serious acidosis results and if untreated or not resolved by the body, coma and death can occur.Most often the acetyl CoA in a type 2 patient is converted to cholesterol and results in hyperlipidemia and heart disease in these patients.The elderly type 2 diabetic is at risk for a hyperosmolar nonketotic coma. The patient becomes dehydrated due to increased urine excretion to lower the blood glucose. If reduced renal or cardiac function is also present, glucose excretion is impaired and blood glucose concentrations can become extremely high. Ketones are not produced in excess, thus the patient remains nonketotic. Insufficient hydration, elevated blood glucose, and decreased renal excretion of waste products result in an increased osmolality and total concentration of all plasma components.

Before glucose meters were available, urine glucose was frequently used to approximate diabetic glucose levels. Blood glucose levels can be related to urine glucose concentration because of urinary excretion of glucose. Physician offices, clinics, and patients at home tested urine with reagent strips for a semi-quantitative measurement of urine glucose and adjustments in insulin therapy were made. Monitoring a diabetic carbohydrate management is seldom performed this way today. Portable meter measurement of blood glucose is a much better management method. Urine glucose measurement is neither sensitive nor specific and does not give information about blood glucose below the renal threshold (usually 180 mg/dL).As a semiquantitative measurement, urine glucose is a routine assay on urinalysis test and an abnormal result would be investigated with blood levels. If quantitative measurements are needed, a timed urine specimen is collected and measured for glucose by blood glucose methods.

Microalbumin is not a measurement of a small size albumin molecule but measurement of low concentrations of urinary albumin in diabetes to identify early renal impairment. Microalbuminuria tests measure concentrations of albumin that are lower than levels that can be detected with routine urine dipstick tests for protein. Timed, overnight, and first morning specimens can be screened for microalbuminuria. Quantitative measurements are also utilized for screening of renal impairment and for monitoring treatment.

Serum and plasma are the most common clinical specimens used for electrophoresis applications. Urine and cerebrospinal fluids (CSF) are also suitable. Other body fluids such as pleural fluid and pericardial fluid are analyzed less frequently. Some specimens require pretreatment before electrophoresis. Low concentrations of proteins normally in urine and CSF are concentrated in order to have enough proteins for detectable separations. Some body fluids require removal of pigments, salts, and other compounds that interfere with electrophoresis or the detection of separated solutes. In molecular diagnostic testing of DNA and RNA, the nucleic acids must first be isolated from the specimen and then purified before separation with electrophoresis.

Routine electrophoresis is a generic term for the traditional clinical laboratory electrophoresis performed on a rectangle-shaped slab gel. Routine electrophoresis is mostly used for separation of proteins and has some use in separating nucleic acids. Generally several patient specimens and control(s) can be placed on one gel and solutes separated in one run. This type of electrophoresis is sometimes called zone electrophoresis.A serum sample with normal plasma proteins yields five zones or bands of separated proteins: albumin, alpha-1-globulins, alpha-2-globulins, beta-globulins, and gamma-globulins. Proteins in CSF and urine proteins are also separated with routine electrophoresis. Using whole blood treated with a reagent to lyse red blood cells, variant and glycosylated hemoglobins can be detected. With different visualization methods, isoenzymes and lipoproteins in a serum sample can be identified.A manual agarose gel electrophoresis of eight serum samples is pictured below. After electrophoresis, the gel was stained with Ponceau S.

With a pH 8.0-9.0 used for protein electrophoresis, proteins take on a negative charge, that is a negative ion cloud forms. As the negative ion cloud migrates to the anode, the proteins are pulled to the anode. Several gels used routinely for protein electrophoresis attract positive ions from the buffer and form a positive ion cloud. This ion cloud moves in the opposite direction to the cathode. This phenomenon is called electroendosmosis or endosmosis.The tension created by these oppositely moving ion clouds can affect the movement of sample macromolecules. The migration of some proteins can be slowed, some proteins can become immobile, and other proteins are pushed toward the cathode. Many protein electrophoresis methods take advantage of this tension and use it to achieve better separation of protein bands. The gamma globulin band in serum, urine, and other body fluids will separate more sharply by being pushed to the cathode and will appear behind the point of sample application.

The typical daily diet of most Americans contains approximately 10 to 15 mg of iron. Sources of dietary iron include heme iron from meats and nonheme iron from whole grains and vegetables. Many processed foods, such as breakfast cereal, are fortified with iron. However, the normal individual absorbs only 5% to 15% of dietary iron, or about 1 to 2 mg daily. Females may absorb slightly more iron than males as they require more iron to replace that lost through menstruation and to meet the increased need for iron in pregnancy.Absorption of iron occurs through the mucosal cells in the duodenum (proximal small intestine). Dietary iron that is not absorbed is excreted in the feces. Intestinal absorption provides the means for regulating the amount of iron in the body.The amount of Iron absorbed is normally low because iron is well conserved within the body. Heme iron from senescent erythrocytes is cycled back into the iron pool and reused for incorporation into developing erythrocytes. Furthermore, iron is normally lost from the body only in very small amounts, primarily through desquamation of mucosal cells in the gastrointestinal tract and losses through body secretions, including urine, sweat and feces. Therefore, under normal conditions, very little dietary iron needs to be absorbed to maintain iron homeostasis.(3)

In addition to the responsibilities listed for Level 3, over 40 laboratories also participate in Level 2 activities. At this level, laboratory personnel are trained to detect exposure to a limited number of toxic chemical agents in human blood or urine, the analysis of cyanide and toxic metals in human samples, for example.

Specialists in immunohematology perform all testing prior to blood transfusions and work to prevent transfusion infections. They also investigate any post-transfusion reactions. This specialty includes all lab procedures performed in the specialty of histocompatibility. Specialists in clinical chemistry analyze body fluids such as blood, urine, and spinal fluid to determine the chemical makeup, including the amount of carbohydrates, proteins, enzymes, and trace elements. The special covers urine microscopics and chemical evaluation of the liver, kidneys, lungs, heart, and other vital organ systems. This specialty also covers all testing performed in the specialties of radioassay and blood gas analysis. Specialists in blood banking can perform all immunohematology testing as well as testing from the specialties of clinical chemistry, hematology and serology/immunology that relates to donor blood. Clinical laboratory personnel who are licensed in the specialties of immunohematology, clinical chemistry, hematology, and serology / immunology may perform all tests in the blood banking specialty.

Do not leave test orders or test results in areas where they can be viewed by patients.Do not discuss test results or any patient information in areas where patients can overhear the conversation. Be careful not to discuss confidential information on the telephone where patients can overhear the conversation.Do not provide supplies to physician offices other than those usually provided by the laboratory. Document any supplies given to an office.Do not supply items that the office can use for testing (e.g. urine dipsticks). Do not allow offices to dispose of biohazard waste or sharps in the waste containers paid for by the laboratory.

To discuss PGx, we must first define two terms - polymorphism and cytochrome P450 (CYP450).A polymorphism is a variation in a gene (allele) that affects at least 1% of the population. CYP450 refers to a family of enzymes found predominantly in the liver. CYP450 enzymes work on a variety of substrates (drugs), altering their chemical structures to facilitate excretion in the urine and feces. There are many known polymorphisms in CYP450 enzymes.

An experienced phlebotomist should be knowledgeable in the collection of: - Venous blood specimens - Capillary blood specimens - Blood culture specimens - Urine specimens - Throat cultures, and - Medicolegal specimens requiring chain of custody. He or she may also need to know how to: - Process specimens - Perform point-of-care tests, and - Collection specimens from IV lines and central venous lines, under appropriate supervision.

If a urine specimen is require, instruct the donor on its collection.Escort him or her to the restroom.The donor breaks the seal on the urine container and produces a specimen.

You may be required to measure the temperature of the urine, and to check it visually for tampering.Apply a tamper-evident seal to the specimen, and label it appropriately in the presence of the donor individual.

The presence of many squamous epithelial cells (SQEs) also indicates a poorly collected urine specimen. If many SQEs are noted upon microscopic examination, the specimen should be recollected. The patient must be instructed how to collect a midstream, clean catch specimen. A Gram stain of a fresh, midstream urine sample would provide information that could help the physician decide whether to prescribe an antibiotic and the choice of antibiotic based on gram-reaction of the bacteria. Examine a Gram-stained slide made from a drop of uncentrifuged urine under oil immersion (1000X) magnification. If more than one bacterial organism is observed per oil immersion field, it can be determined that the quantity of bacteria is >105 colony forming units (CFU) per mL, and the patient probably has a urinary tract infection (UTI). The Gram stain reaction would also be important. Most bacteria that cause UTIs are gram-negative Enterobacteriaceae. A Gram stain report in this case would be "gram-negative bacilli consistent with quantity >105 CFU/mL."

Epithelial cells in large numbers within sputum smears means that the specimen is predominantly oral saliva, rather than true sputum from the lung. Epithelial cells in urine smears indicate that the sample has been contaminated by organisms found on the vulva or distal urethra. Bacteria found near or on epithelial cells are usually normal contaminating bacterial flora.White blood cells indicate inflammation and possible infection. The direct smear examination should focus within and around these cells.Red blood cells in a direct smear are not usually significant.Yeast may be present as normal flora in upper respiratory tract or genital tract. They may be significant if they predominate, or if budding yeast forms are seen.Hyphae are more likely to indicate the presence of fungal infection, but this determination requires correlation with clinical findings.Bacteria found in spinal fluid, blood, tissue and specimens from other sterile sites are always significant.Body fluids which are normally sterile must be examined carefully. If only one organism per oil immersion field is identified, then there are about 105 organisms per mL present in the sample! Bacteria observed in specimens from the throat, genital tract and other areas containing normal flora suggest infection only if their composition and type varies significantly from the norm.

A study that was published in 2002 concluded that 68 - 87% of laboratory errors occur in the preanalytic and postanalytic stages of the testing process with the majority occurring in the preanalytic phase.* Steps in the pre-analytic phase occur both inside and outside the laboratory, and are performed by both laboratory and non-laboratory personnel. While the following list is not exhaustive, some of the most common sources of error in the preanalytic phase include:Patient preparation Patient not told to be fasting; improper or no instruction to patient on proper collection of specimen such as clean catch urine. Patient injured during phlebotomy Development of hematoma resulting in no specimen obtained for testing. Requisition errors Patient information missing, illegible, or on wrong patient; wrong tests ordered. Patient identification Patient incorrectly identified. Labeling of specimen Specimen not labeled or incorrectly labeled. Preparation of specimen Specimen centrifuged too long or not long enough; specimen placed in improper preservative.Storage of specimen Specimen not refrigerated or frozen as required or refrigerated when it should be at ambient temperature. Shipment of specimen Shipped at ambient temperature when it should have been shipped frozen; delay in shipment. Accessioning process including preparation for analysis Sorted into wrong batch; incorrect labeling. Order entry Incorrect data entered during manual entry of a test requisition. Specimen sub-optimal Not enough specimen for testing; visible hemolysis. Contamination Inadequate cleansing of venipuncture site resulting in contamination during blood culture collection. *Reference: Bonini P, Plebani M, Ceriotti F, Rubboli F. Errors in laboratory medicine. Clin Chem. 2002;48:691-698. Available at http://www.clinchem.org/cgi/content/full/48/5/691#T2BAccessed June 23, 2010.

Glucose tolerance tests are used to help diagnose diabetes mellitus or gestational diabetes, which occurs during pregnancy. The procedure basically consists of these steps:Confirm that the patient has been fasting.Collect a fasting blood glucose specimen. Have the patient drink the dose of glucose solution required by the procedure.Collect blood at standard timed intervals. Blood, or blood and urine specimens, are then checked for glucose levels. The patient should be instructed to remain in the facility and remain seated between blood collections. The phlebotomist should check on the patient periodically between blood collections, especially during the first hour. For some patients, the glucose solution may cause nausea and vomiting and the test may need to be terminated.

The following factors promote the formation of casts in the kidney:Larger than normal amounts of plasma proteins entering the tubulesDecreased pHDecreased urinary flow rateIncreased urine concentrationAfter formation, casts are loosened from the tubules and discharged into the urine.

The urine microscopic exam is performed on a centrifuged urine sediment. The sediment contains all the formed elements or insoluble materials that have accumulated in the urine through its passage from the kidney to the lower urinary tract. These formed elements include cells, casts, crystals, and miscellaneous structures.

The microscopic examination was traditionally performed on all urine specimens. Today, many laboratories perform a urine microscopic only if preliminary evaluation indicates the need for microscopic examination. Such laboratories must have criteria determining the specimens on which urine microscopic examinations will be performed. The microscopic exam is often important in detecting and evaluating renal and urinary tract disorders as well as other systemic diseases.

Examination of a fresh urine specimen provides the best results. Bacteria will metabolize glucose and pH changes may occur, affecting these results that are reported as part of the macroscopic urine testing. If the specimen is dilute (specific gravity <1.010), and/or the pH is >7.0, casts, white blood cells and red blood cells may lyse. If an unpreserved specimen (i.e., specimen container does not contain a preservative) cannot be tested within two hours of collection, it should be refrigerated at 2°-8° C. Although the most commonly received urine specimen is the random urine collection, the specimen of choice for urinalysis is the first morning urine. The first morning urine is more concentrated and allows for the detection of substances, which may not be present in a more dilute random sample. Once the physical and chemical characteristics of the urine have been determined, the microscopic exam is performed on the sediment.

Increased numbers of cuboidal cells are found in renal transplant rejection, acute tubular necrosis (diuretic phase), injuries that interrupt blood flow to the kidney, and acute glomerulonephritis accompanied by tubular damage. Ingestion of various drugs and chemicals may cause significant tubular shedding of these epithelial cells. Cuboidal cells are easily seen in urine in cases of salicylate intoxication.

In contrast, RBCs appear swollen in dilute or alkaline urine, having taken on water from their surroundings.

The most common type of cell seen in the urine sediment is the epithelial cell. This slide shows squamous epithelial cells under low power brightfield microscopy (upper image) and a squamous epithelial cell under high power. The sediment in the lower image was stained with a supravital stain. The cells appear as large flattened cells with abundant cytoplasm and small round central nucleus. Although squamous epithelial cells have little clinical significance they must be differentiated from other cellular elements.

Cells which may be present in the urine include epithelial cells, white blood cells (WBCs) and red blood cells (RBCs). The epithelial cells in the urine may originate from any site in the genitourinary tract. It is normal to find a few epithelial cells in the sediment. WBCs may enter the urinary tract anywhere from the glomerulus to the urethra. The WBCs are mostly neutrophils. RBCs may originate in any part of the urinary tract. Normally, RBCs do not appear in the urine, although the presence of a few RBCs is not considered abnormal.

Tyrosine crystals appear as fine silky needles arranged in sheaves or bundles in acid urine. They are rarely present and may appear together with leucine crystals in liver disease. Do not confuse tyrosine with crystals caused by x-ray dye. X-ray dyes will cause the urine specific gravity to be greatly increased (1.040), Tyrosine crystals are soluble in alkali or dilute mineral acid.

Cholesterol crystals may be seen in renal tubular disease. These crystals look like plates of glass, sometimes with a notch out of one corner. Under polarized light, they exhibit a stained glass effect. These crystals are rarely seen unless the specimen has been refrigerated, because the lipids remain in droplet form. Large amounts of protein, lipid droplets, fatty casts or oval fat bodies should be found along with cholesterol crystals. Cholesterol crystals are found in acid or neutral urine.

Bilirubin crystals are seen in the urine when the serum bilirubin level is increased. The macroscopic appearance of urine with bilirubin crystals is orange to almost black in color. The crystals themselves appear as gold-orange needle-like forms that are sometimes clumped together.

Crystals are not usually present in freshly voided urine, but can appear in urine left at room temperature for several hours. Most crystals form due to changes in urine pH and temperature after collection. Diagnostically significant crystals may indicate the presence of a metabolic disorder, renal calculi formation, or provide information that can be used to regulate medications.

The following table lists common crystals found in the urine sediment. Crystals that have no clinical significance must be identified and differentiated from those that can be an indication of a metabolic disorder or other clinically significant conditions.CrystalpHColorUric acidAcidicYellow - brownCalcium oxalateAcidic/neutralColorlessAmorphous uratesAcidicYellow - brownTriple phosphateAlkalineColorlessAmmonium biurateAlkalineYellow - brownAmorphous phosphateAlkaline/neutralWhite - colorlessCalcium carbonateAlkalineColorless

Crystals found in normal acid urine include uric acid, calcium oxalate and amorphous urates. This slide shows an example of uric acid crystals.

Ammonium biurate crystals commonly occur in the form of "thorn apples," as shown here, or in polyhedral shapes. They are deeply colored from a dark yellow to brown. They sometimes appear in clumps or clusters. This crystal occurs only in stale urine.

In order for a urinalysis to be useful a physician must know not only what elements are present but the quantity of each. This section will deal with counting and estimating the microscopic elements found in the urine sediment. The quantifications may vary slightly between laboratories, but each lab should have its own criteria. Quantitation may be divided into three steps: Looking for casts Counting elements Estimating elements

Patients with hypersensitivity reactions, sometimes as a result of medications, may have eosinophils in their urine. Hansel's stain, which is specific for eosinophils, or Wright-Giemsa stain may be used to distinguish these cells from neutrophils.

Another parasite which may be seen in urine is the Enterobius vermicularis (pinworm) ova. The ovum is ovoid in shape with one side flattened. The embryo is separated from the shell by a clear space.

When the urine cools or when the wet mount begins to dry, Trichomonas begins to lose its characteristic motility and may easily be misinterpreted as white cell or epithelial cell.

Sperm may be present in urine sediment. Sperm have a characteristic oval body with a long thin tail and are 50 microns in length.

A unit of red blood cells (RBCs) contained 250 mg of iron as part of the hemoglobin molecule. A long-term complication of red cell transfusion is iron overload, or hemosiderosis. As red cells are destroyed, they release iron. The iron cannot be excreted and is stored as hemosiderin and ferritin. Iron accumulates in the liver, heart, spleen, and endocrine organs. Tissue damage, heart failure, liver failure, diabetes, and hypothyroidism can occur. Patients who are transfused frequently are at the greatest risk for iron overload. Diseases such as sickle cell disease, thalessemia, aplastic anemia, and other chronic anemias usually require frequent transfusions. Signs and symptoms of hemosiderosis include muscle weakness, fatigue, weight loss, mild jaundice, anemia, and cardiac arrhythmias. Ferritin levels and other iron studies should be assessed. Specific stains may be used to detect iron in tissue biopsies. Iron chelation may be used to treat and prevent iron overload. Chelation works by using an agent which binds to iron and helps remove it through the urine or feces.

If preliminary testing suggests hemolysis or if the results are misleading, additional testing may be required. If human error has been ruled out during the clerical check, repeat ABO/Rh testing should be performed on the unit of blood or its segment and the pre-transfusion sample to detect any sample mix ups and clerical errors. Antibody detection studies should be performed on the pre- and post-transfusion samples to look for any unidentified antibodies. If an antibody is identified, the donor cells should be tested for the corresponding antigen. The crossmatch should be repeated with pre-and post-tranfusion specimens using the indirect antiglobulin test (IAT). An incompatible crossmatch with the pre-transfusion sample indicates an original error, either clerical or technical. Incompatibility with only the post-transfusion sample indicates a possible anamnestic response, as in a delayed hemolytic transfusion reaction (DHTR), or sample misidentification. The patient's first voided urine specimen should be examined for the presence of free hemoglobin. The patient's bilirubin levels may also be evaluated. A change from normal pale yellow serum to a post-transfusion bright or deep yellow serum should prompt an investigation for hemolysis. The maximum concentration of bilirubin following hemolysis is not usually detectable until 3 to 6 hours after transfusion. The hemoglobin and hematocrit can be tested to detect a drop in hemoglobin or failure of the hemoglobin to rise after transfusion. Important information about physical or chemical hemolysis may be gained from examining the returned unit bag. If hemolysis is present in the bag or tubing, a process which affected the blood, such as inappropriate warming or faulty infusion pump, should be suspected. If bacterial contamination is suspected, the unit can be cultured. A positive culture indicates a reaction due to bacterial contamination.

Although there is no consistent clinical picture of an acute hemolytic transfussion reaction (AHTR), common symptoms include chills, hypotension, and fever. Some patients have experienced pain at the infusion site, flank pain, and anxiety with a feeling of doom. Red or dark urine may be the first sign of intravascular hemolysis. If patients are unconscious or in surgery, changes in vital signs, unexplained bleeding, or hemoglobinuria may be the only signs. Additional signs and symptoms include, but are not limited to: rigors, facial flushing, chest and abdominal pain, nausea and vomiting, dyspnea, oliguria/anuria, diffuse bleeding, shock, and renal failure. The severity of symptoms is related to the amount of incompatible blood transfused. Patients with underlying diseases that involve intravascular hemolysis can make diagnosis extremely difficult.

Plasma cells are uncommonly observed in the peripheral blood smear. They are normal constituents of lymph nodes, spleen, connective tissue and bone marrow. The presence of plasma cells in the peripheral blood is indicative of a large number of conditions, mostly related to infections , immune disorders, malignancies, toxic exposures, hypersensitivity reactions and their responses.Although mature plasma cells have a distinct appearance, they still may be confused morphologically with immature plasma cells and other cells with inclusions, reactive changes or nucleated red bloods cell with altered identities. In the image to the right, a plasma cell is present. The plasma cell has an eccentric immature nucleus with a muddy chromatin pattern. Note also clumping and stacking of the erythrocytes, typical of rouleaux formation, implicating an increase in plasma gamma globulin. Further studies are in order, including a bone marrow examination, where at least 30% of bone marrow cells should be variations of mature and immature plasma cells. Serum protein electrophoresis will reveal a monoclonal globulin spike, and light chains in excess of 1.0 gm/24 hours may be seen in the urine. The presence of lytic bone lesions is a convincing clinical clue. With these findings in combination, a diagnosis of myeloma can be made with assurance.