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Urine Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Urine and links to relevant pages within the course.

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Advances in Noninvasive Prenatal Testing For Down Syndrome and other Trisomies
Screening Tests, continued: Maternal Serum Screening

Maternal serum screening tests may be performed during the first and second trimester of pregnancy. These serum tests involve the measurement of specific biomarkers in the maternal blood. These five biomarkers are typically used to assess the risk for Down syndrome: Maternal Serum Alpha-fetoprotein (MS-AFP) AFP is a fetal protein that is initially produced in the fetal yolk sac and liver; by the end of the first trimester of pregnancy, most if not all of the AFP is produced by the fetal liver. The concentration of AFP peaks in fetal serum at 10-13 weeks gestation. The fetal AFP normally diffuses across the placental barrier and into the maternal circulation so that MS-AFP rises throughout pregnancy to about 250 ng/mL at 32 weeks gestation. Lower MS-AFP values may be associated with increased risk for trisomy 21 (Down syndrome) or trisomy 18 (Edwards syndrome).Unconjugated estriol (uE3-estriol) Estriol is a female sex hormone that increases during pregnancy. It is produced by the placenta and passes into the maternal bloodstream during pregnancy. Its roles during pregnancy may be linked to the proper functioning of the uterus, softening of the cervix, and assistance in the lactation process. A biologically active form of estriol called uE3-estriol increases in the maternal circulation during pregnancy by the seventh to ninth weeks of gestation and continues to increase throughout pregnancy. uE3-estriol levels in maternal serum is typically decreased in the Down syndrome pregnancy. Human chorionic gonadotropin, total or free beta subunit (beta-hCG) HCG is a glycoprotein hormone produced by the placenta during pregnancy. It is present in blood and urine around 7-13 days following fertilization of the ovum. HCG has two subunit chains, alpha and beta. The beta subunit confers its specificity. A specific smaller part of the hormone, called free beta hCG may be used as a screening test during the first trimester of pregnancy. An increase in maternal serum free beta hCG (ie, greater concentration than in other pregnancies) may indicate an increased risk for Down syndrome. Total beta-hCG is tested as part of a triple or quad screen during the second trimester of pregnancy. An increase in total beta hCG in the maternal serum is also associated with increased risk of Down syndrome.Dimeric inhibin A (DIA) Inhibins are a family of glycoproteins mainly secreted by the ovaries and testicles. The beta subunits of the inhibins exist in two forms, the A and B forms. DIA is secreted by the ovaries and is designed to inhibit the production of the hormone FSH by the pituitary gland. The level of DIA is increased in the blood of mothers of fetuses with Down syndromePregnancy Associated Plasma Protein A (PAPP-A) PAPP-A is produced by the covering of the newly fertilized egg. It is thought to be involved in local proliferative processes such as wound healing and bone remodeling. Unexplained low levels of PAPP-A in the maternal serum during pregnancy may indicate increased chance for intrauterine growth restrictions, premature delivery, preeclampsia, and stillbirth. In the first trimester, low levels of this protein are seen in Down syndrome pregnancies.In the first trimester, serum screening is typically done in combination with an ultrasound to screen for nuchal translucency. Serum screening in the first trimester usually involves the measurement of two biomarkers in the maternal serum, free beta-hCG and PAPP-A. Combining the results of these two biomarkers with an ultrasound improves the screening process. In the second trimester during weeks 16-18, maternal serum assays for 3-4 levels of biomarkers are typically performed. The screening for these biomarkers has been established as a triple or quadruple (quad) screen, since the benefit of the screening lies in the combined use of the three to four biomarkers. The biomarkers used in the screening process may include MS-AFP, uE3-estriol, total beta HCG, and DIA. Increased serum levels of MS-hCG and DIA combined with decreased levels of UE3-estriol and MS-AFP suggests an increased risk of Down syndrome. Maternal age, family history, weight, race, and diabetic status are also used to determine a numeric risk for Down syndrome. It is important to understand that for women who have positive triple or quad screening results, only a very small number of them have babies who actually have a chromosomal abnormality.

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Blood Banking Question Bank - Review Mode (no CE)
An urticarial reaction is characterized by:View Page

Case Studies in Clinical Microbiology
Clinical History

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.

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The bacterial species shown growing on 5% sheep blood agar was recovered from the spun sediment of a midstream urine specimen after 24 hours incubation at 35C. Each of the following tests would be useful in supporting the presumptive identification of Enterococcus species except: (Choose all that apply)View Page

Chemical Screening of Urine by Reagent Strip
Chemical Urinalysis Reagent Strips

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.

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Precautions in Urinalysis Chemical Reagent Strip Analysis

The following precautions should be observed when working with urinalysis reagent strips: Store strips according to the manufacturer's recommendation.DO NOT expose strips to moisture, volatile fumes, or direct sunlight. 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 or 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.Procedural PrecautionsAlthough the procedure is simple to perform, accurate results depend on careful adherence to manufacturer's directions and adequate quality control.Normal and abnormal controls should be tested whenever a new lot of strips is opened, and at the frequency defined by the laboratory's procedure.If quality control results do not correspond to the published control values, the problem must be resolved before patient samples are tested.High levels of ascorbic acid (Vitamin C) in the urine may inhibit some reagent strip reactions, such as glucose, blood, bilirubin, nitrate and leukocyte esterase. The urine dipstick's package insert will provide information about potential interfering substances, including ascorbic acid.Intensely colored urine may make it difficult to correctly interpret color reactions on the dipstick, as illustrated below. The affected tests should not be reported from the dipstick. It would be necessary to use an alternative method of testing if available.

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Urine Specimen Collection

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.

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Reagent Strip Procedure

Using 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 towel or gauze to remove excess urine (do not blot the strip). 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. Each laboratory may have a slightly different urinalysis procedure for manual chemical reagent strips. However, the procedure below is a general version that should apply to most laboratories.Maintain the strip in a horizontal position to prevent mixing of reagent chemicals and observe the reagent pads at the specified time periods as indicated on the chemical reagent strip container or procedure. 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.Carefully record results on pre-printed worksheets upon observation of each reagent pad result.The video on this page demonstrates correct technique when a dipstick reader is used.

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All the following statements about the urine specimen are true EXCEPT:View Page
Urine Specimen Processing and Transportation

In order to ensure proper stability of the specimen and accurate test results, there are guidelines in place to aid in the appropriate urine processing and transportation. These guidelines include: Ensuring that all urine collection and/or transport containers should be clean and free of debris or interfering substances.Ensuring that the collection and/or transport container has a secure lid and is leak resistant. Leak-resistant containers reduce specimen loss and healthcare worker exposure to the specimen while also protecting the specimen from contaminants.Utilizing urine containers that are made of break-resistant plastic instead of glass.Utilizing urine containers that do not leach interfering substances into the specimen.Utilizing collection containers and/or transport tubes which will not leak within the pneumatic tube system (if one is used within the laboratory facility). A leak-proof device in this situation is paramount.Proper labeling and correct identification should be applied to the collection container or tubes. This includes noting the time the specimen was collected. Remember that urinalysis specimens must be analyzed within 2 hours of collection.Ensuring that there is sufficient volume to fill the tubes and/or perform the test.

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Basis of the Urine pH Test

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.

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Urine pH: Acidic and Alkaline

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 functionCrystal or calculi formationDietThe table below summarizes dietary and medical conditions as well as preanalytic and analytic errors 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)XKidney failureXUrinary tract infectionsXVomitingXDiabetic ketoacidosis XDiarrheaXStarvationX

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The indicator(s) used in the pH test region of the chemical reagent strips for urine is/are: (Choose ALL correct answers)View Page
A urine specimen was collected at 6:00 a.m. and remained at room temperature until it was received in the laboratory at 3:30 p.m. How may the pH of the specimen be affected by the extended time at room temperature if bacteria are present in the specimen?View Page
Match the following factors with the expected urine pH type:View Page
pH Analysis: The Urine Specimen

The urine specimen should be freshly voided. Urine is an ideal medium for the proliferation of bacteria due to the large amount of urea present. These bacteria metabolize urea, producing ammonia that causes the urine pH to become more alkaline. If there is a delay before performance of the test, the sample should be refrigerated.

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Protein Error of Indicators

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.

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Follow-up Testing for Positive Urine Protein

A 24-hour urine protein may be ordered if a large amount of protein is detected with the dipstick method or if protein persists in the urine. A 24-hour urine protein may also be ordered if the physician suspects the release into the urine of protein other than albumin.

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Confirmatory Testing for Urine Protein

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.

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Clinical Significance of Urine Protein

The presence of an increased amount of protein in a urine specimen is often the first indicator of renal disease. Proteinuria may signal severe kidney damage, be a warning of impending kidney involvement, or be transient and unrelated to the renal system. Further quantitative testing of urine for protein may be needed to determine the significance of the 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.

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Clinical Significance of Urine Protein (continued)

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.

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The presence of increased levels of protein in the urine may be an early indicator of which of the following conditions?View Page
Examples of conditions resulting in benign proteinuria include: (Choose ALL correct answers)View Page
Urine Glucose Analysis

The analysis for glucose on a chemical reagent strip is a double-sequential enzyme reaction, utilizing the glucose-oxidase/peroxidase method. In the first reaction, glucose oxidase catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide. Then, the peroxidase catalyzes the oxidation of a chromogen by the hydrogen peroxide to form a colored product. The chemical reagent strip glucose pad is then analyzed and recorded at the set interval stated by the manufacturer.This method does not react with lactose, fructose or galactose. Study the dipstick color chart in your laboratory to become familiar with the range of color changes. NOTE: The urine specimen should be analyzed while at room temperature for these enzyme reactions to occur properly.

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False Positive and Negative Urine Glucose Results

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.

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Test for Reducing Substances Other than Glucose

Urine specimens from certain pediatric patients should be tested for other reducing substances, such as galactose, when the results for glucose are negative using the routine chemical reagent strip method. The laboratory's procedure should define when additional testing is needed. For example, some laboratories will peform this additional step for each urine specimen received from patients two years old or younger.

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Clinical Significance of Glucose in the Urine

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

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Which of the following conditions produce glycosuria? (Choose ALL correct answers)View Page
Ketones Overview

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.

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Testing for Ketones in the Urine

Testing for ketone bodies is based on a nitroprusside reaction. Acetoacetic acid reacts with sodium nitroferricyanide and glycine in an alkaline medium to produce a violet-to-purple colored complex. The urine chemical reagent strip method can detect as little as 5 mg/dL acetoacetic acid in urine. It does not react with acetone unless glycine is present or B-hydroxybutyric acid. Since these two compounds are derived from acetoacetic acid, their presence can be assumed if the test for ketones is positive. Ketones are reported either as negative, small, moderate or large amounts; or negative, 1+, 2+, 3+, or 4+. In some severe cases of ketosis, it may be necessary to perform tests on serial dilutions to provide more information on the quantity of ketones present.

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Ketone Bodies

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.

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False Positive and Negative Ketone Results

False Positive KetonesFalse positive ketone results may be seen in patients after BSP or PSP dye injection due to the phthaleins. The presence of L-DOPA metabolites, some urine preservatives (e.g. 8-hydroxyquinaline), or high levels of phenylketones will also cause false positive results. Antihypertensive drugs such as methyldopa and captopril also may produce false positive results.False Negative KetonesThe 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.

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Clinical Significance of Positive Urine Ketones

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.

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The ketone component that is measured by the nitroprusside reaction is:View Page
Which of the following can cause a false positive result for ketones? (Choose ALL correct answers)View Page
Significantly increased levels of ketones are detected in the urine with which of the following conditions? (Choose ALL correct answers)View Page
Overview of Bilirubin

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.

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Testing for Urine Bilirubin

The test for bilirubin on the urine chemical reagent strip is based on the formation of an azobilirubin compound resulting from a reaction of bilirubin in an acid medium with diazotized 2, 4 dichloroaniline. The color of this compound ranges through various shades of tan. Some sources describe the colors produced as shades of tan-to-pink-to-violet.Since other pigments in the urine may influence the test results, this test strip is more difficult to interpret than the others. Colors which are unlike either the positive or negative color blocks on the color chart may be due to the presence of bilirubin -derived bile pigments. Any urine which demonstrates an atypical color on the bilirubin test strip should be tested further. Even a slight change in color should be considered significant since bilirubin is never present in normal urine.

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False Positive and Negative Urine Bilirubin Results

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.

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Confirmatory Testing for Urine Bilirubin

Confirmatory testing using an alternative method, such as Ictotest® reagent tablets, can be performed when positive results are seen on the dipstick strip, when a red color forms on the strip, or when atypical color changes occur that are the result of bilirubin-derived bile pigments in the urine masking the bilirubin reaction. The Ictotest® employs the same diazotization reaction as the reagent strip, but should not give a false positive result with colored urines.

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Clinical Significance of Urine Bilirubin

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.

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Which of the following may cause a false positive bilirubin result on a urine chemical reagent strip?View Page
When a patient has a bile duct obstruction, the bilirubin test portion of the reagent strip is:View Page
Introduction to Hematuria

The term hematuria is used to describe the presence of intact red blood cells in the urine. The urine may be cloudy/red or pink in color and red blood cells are visible upon microscopic examination.If the red blood cells have been destroyed, hemoglobin will be excreted in the urine. The term, hemoglobinuria, is used to describe this condition. The color of the urine will be pink or red but clear rather than cloudy. The presence of only five red blood cells per microliter of urine is considered to be clinically significant. For this reason, a chemical test is needed to detect quantities of blood too small to change the color of the urine. Microscopic examination is used to differentiate between hematuria and hemoglobinuria if the chemical reagent strip is positive for blood.

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Reagent Test Pad for Blood

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.

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False Positive and Negative Results

False Positives:A false positive result for blood on the urine chemical reagent strip can occur when oxidizing contaminants, such as hypochlorite (bleach), remain in collection bottles after cleaning. Contamination of the urine with provodine-iodine, a strong oxidizing agent, used in surgical procedures can also result in a false positive reaction. Microbial peroxide found in association with urinary tract infections may also cause false-positive results. Capoten® (Captopril) can cause decreased reactivity.The muscle tissue form of hemoglobin, myoglobin is a well-known cause of false-positive reactions on the blood portion of the reagent strip. When tissue hemoglobin is present, the urine specimen has a clear red appearance. Patients suffering from muscle-wasting disorders or muscular destruction due to trauma, prolonged coma, or convulsions or individuals engaging in extensive exertion may have myoglobin in their urine. Specific tests for myoglobin, such as immunodiffusion techniques or protein electrophoresis, are needed to confirm the presence of this substance in a urine specimen. Levels of ascorbic acid normally found in urine do not interfere with this test. False Negatives:False negative results may occur in some analysis 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.

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Clinical Significance of Blood in 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

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A urine sample is cloudy pink in appearance. The microscopic examination reveals the presence of intact red blood cells. The term used to describe these findings is:View Page
Which of the following substances can cause a false positive result for blood on the urine chemical reagent strip?View Page
Match the following positive urine blood results with the possible conditions which cause them to occur.View Page
Urine Analysis for Nitrites

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.

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Nitrites and Bladder Infections

Bladder infections are usually caused by gram-negative bacteria. These bacteria reduce nitrates to nitrites when urine remains in the bladder three to four hours. 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.

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Nitrite Test Sensitivity

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 nitrates.

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False Positive and Negative Results

False Positives: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. False Negatives:False negative results may occur in urine specimens that did not remain in the bladder for the sufficient length of time needed for the bacteria to reduce a measurable quantity of nitrate to nitrite. Other reasons for false negative results include high specific gravity, ascorbic acid levels above 25mg/dL or a low pH (<6). Less frequently, the cause may be due to a lack of sufficient nitrate in the diet (green vegetables) or further reduction of nitrite to nitrogen when large numbers of bacteria are present. In patients receiving antibiotics, the metabolism of the bacteria may be inhibited which would also produce a false negative reaction.

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Clinical Significance of Nitrites in Urine

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.

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Nitrates in urine are reduce to nitrites by:View Page
The nitrite portion of the test strip can be used to: (Choose ALL correct answers)View Page
Introduction to Urobilinogen

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 levels of urobilinogen and the patient should be evaluated further. It is important to note that the chemical reagent strip cannot determine the absence of urobilinogen, so a negative result is impossible.

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Urobilinogen Analysis

The test for urobilinogen 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.

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False-Positive and False-Negative Urobilinogen Results

False-positive results:A false positive urobilinogen reaction may occur with the chemical reagent strip method when substances known to react with Ehrlich's reagent such as sulfonamides and p-aminosalicylic acid are present in the urine. Drugs that contain Azo dyes, such as Azo Gantrisin®, have a gold color that masks the reaction, causing a false positive reaction. Atypical color reactions may be obtained in the presence of high concentrations of p-aminobenzoic acid. The chemical reagent strip urobilinogen test cannot detect porphobilinogen in a urine specimen. Porphobilinogen is a molecule formed during the synthesis of the heme portion of hemoglobin. False-negative resultsDue to the instability of urobilinogen, a false negative result may occur using the chemical reagent strip method if the urine specimen has remained at room temperature for an extended period of time exposed to light. A false negative result may also occur if formalin is present.

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Clinical Significance of Urobilinogen in Urine

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.

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A test that can be used as a confirmatory test for urobilinogen is:View Page
In hemolytic disease, the urine bilirubin test result is negative and the urine urobilinogen test result is:View Page
Presence of Granulocytes in Urine

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 urine chemical reagent strip reaction where indoxylcarbonic acid ester releases 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.

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Leukocyte Esterase on the Urine Chemical Reagent Strip

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.

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False Positive and Negative Urine Leukocyte Esterase

False Positives: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. False Negatives:False negative results may occur in the presence of significant levels of protein or glucose and in urines with high specific gravity which can crenate the white blood cells, leaving them unable to release esterases. Some drugs such as Cephalexin (Kelfex®), Cephalothin Keflin®), Tetracycline, or high concentrations of oxalic acid may also cause falsely decrease leukocyte esterase test results.

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Clinical Significance of Leukocyte Esterase in Urine

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.

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A positive leukocyte esterase test indicates the presence in a urine specimen of which of the following?View Page
Measuring Specific Gravity

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.

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Advantages and Limitations of the Chemical Reagent Strip Method for Specific Gravity

Specific gravity (SG) measured with the chemical reagent strip method correlates well with gravimetric measurement, and, unlike the gravimetric or refractometer methods, does not need to be corrected for glucose or protein. Cloudy/turbid urines do not need to be clarified before measuring specific gravity with the reagent strip method. It is the recommended method for determining specific gravity if a urine specimen contains x-ray contrast media or plasma expanders. Alkaline urine can affect the indicator system and lower the specific gravity result on the reagent pad. If the result is being read visually, it is recommended that .005 be added to the specific gravity result when the pH is alkaline. Most chemical reagent strip readers, however, will automatically adjust the specific gravity reading for pH. A specific gravity reading higher than the chemical reagent strip range would need to be measured by another method, and may require dilution.

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Clinical Significance of 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.

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How does ion concentration in the urine relate to specific gravity?View Page
Semi-automated and automated urine chemical reagent strip readers:View Page
Urine chemical reagent strips readers can utilize any manufacturers dipsticks.View Page
When an automated or semi-automated method is used to read urine chemical reagent strips, quality control testing must be performed at least how often?View Page
Quality Control

Both a normal and an abnormal urine quality control must be tested with each new lot of chemical reagent strips, and at least every day that patient testing occurs in order to confirm the accuracy of the reagent strips and the automated reader. Some automated readers also require periodic calibration. Follow the manufacturer's instructions for calibration procedure and frequency. Quality control results must be recorded, and corrective action must be taken when the results do not fall within the acceptable ranges.

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Chemical Screening of Urine by Reagent Strip (retired March 2012)
Urine should be at room temperature prior to testing with the reagent strip method.View Page
Which of the following statements are TRUE regarding the reagent strip test procedure? (Choose ALL of the correct answers)View Page
A patient that is on a vegetarian diet will most likely have an acid urine pH.View Page
False-positive tests for protein on a urine reagent strip may be caused by: (Choose ALL of the correct answers)View Page
Which of the following tests included on a urine reagent strip would never be reported out as negative?View Page
What effect may bacterial contamination have on urine pH?View Page
Excessive carbohydrate loss that may occur due to vomiting, or rapid weight loss may result in the presence of which of following substances not normally contained in the urine?View Page
A patient suspected of a urinary tract infection has a negative nitrite test, but bacteria is present upon microscopic examination. What may have caused this discrepant result? (Choose ALL of the correct answers)View Page
Which of the following statements are TRUE for specific gravity measured by the reagent strip method? (Select ALL that apply)View Page
When the glucose result on a urine specimen from an infant is negative on the reagent strip, it can be assumed that the specimen is negative for other reducing substances such as galactose.View Page
Sulfosalicylic acid can be used to confirm the result of which of the following tests included on a urine reagent strip?View Page
A voided urine specimen is delivered from the women's clinic to the laboratory six hours after collection. The following results are reported:Color: yellow Protein: negative Bilirubin: negativeClarity: cloudy Glucose: negative Urobilinogen: 0.2 mg/dLSp. Gravity: 1.020 Ketone: negative Nitrite: positivepH: 9.0 Blood: negative Leukocyte esterase: negativeWhat might these results indicate?View Page
Chemical Reagent Strip

A chemical reagent strip for screening urine is a narrow band of paper, which has been saturated with chemical indicators for specific substances or properties at various locations on the strip. The position of the test area may vary depending on the brand and type of strip used. Always read the package insert for specific directions.Notice the relative positions of the test areas and the name of the test that corresponds to each area. The strip should be handled only at the opposite end from the test areas.

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Reagent Strip Precautions

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.

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Specimen Collection, Storage, and Handling

The urine specimen should be collected in a clean container and examined as soon as possible after voiding. If testing will be delayed more than two hours after specimen collection, the specimen should be refrigerated. Allow the specimen to return to room temperature before testing.

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Urine Reagent Strip Procedure

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.

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Procedural Considerations

Although the procedure is simple to perform, accurate results depend on careful adherence to manufacturer's directions and adequate quality control. Normal and abnormal controls should be tested whenever a new lot of strips is opened, and at the frequency defined by the laboratory's procedure. If quality control results do not correspond to the published control values, the problem must be resolved before patient samples are tested. High levels of ascorbic acid (Vitamin C) in the urine may inhibit some reagent strip reactions, such as glucose, blood, bilirubin, nitrate and leukocyte esterase. The urine dipstick's package insert will provide information about potential interfering substances, including ascorbic acid. Intensely colored urine may make it difficult to correctly interpret color reactions on the dipstick, as demonstrated in the image on the right. The affected tests should not be reported from the dipstick. It would be necessary to use an alternative method of testing if available.

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All the following statements about the urine specimen are true EXCEPT:View Page
Which of the following statements are true for the reagent strip procedure? (Choose ALL that apply)View Page
Basis of the Urine pH Test

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.

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pH Value

Urine pH results must be evaluated in conjunction with a patient's medical condition. Factors to be considered include:respiratory or metabolic acidosis respiratory or metabolic alkalosis renal function crystal or calculi formation urinary tract status diet

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Acid and Alkaline Urine pH

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

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Urine Specimen

The urine specimen should be freshly voided. Urine is an ideal medium for the proliferation of bacteria due to the large amount of urea present. These bacteria metabolize urea, producing ammonia that causes the urine pH to become more alkaline. If there is a delay before performance of the test, the sample should be refrigerated.

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The pH range that can be determined on the urine reagent strip is:View Page
A urine specimen was collected at 6:00 A.M. and remained at room temperature until it was received in the laboratory at 3:30 P.M. How may the pH of the specimen be affected by the extended time at room temperature if bacteria are present in the specimen?View Page
Match the following factors with the expected pH:View Page
Protein Error of Indicators

Testing for protein 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 (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 dipstick method for protein.

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False Positive Protein Results

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.

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Follow-up Testing of Urine Dipstick Protein Results

A 24-hour urine protein may be ordered if a large amount of protein is detected with the dipstick method or if protein persists in the urine. A 24-hour urine protein may also be ordered if the physician suspects the release into the urine of protein other than albumin.

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Confirmatory Testing for Protein

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.

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Clinical Significance

The presence of protein in a urine specimen can have serious implications. It may signal severe kidney damage, be a warning of impending kidney involvement, or be transient and unrelated to the renal system. Further quantitative testing of urine for protein may be needed to determine the significance of the proteinuria.

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Clinical Significance cont'd

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.

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Clinical Significance cont'd

Individuals with diabetes mellitus may excrete small amounts of protein in the urine which may signal the beginning of reduced glomerular filtration. Stabilizing the blood glucose level at this time may delay progression of diabetic nephropathy. 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.

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Clinical Significance cont'd

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.

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Glucose Test

The test for glucose is a double sequential enzyme reaction, utilizing the glucose-oxidase/peroxidase method. In the first reaction, glucose oxidase catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide. Then, the peroxidase catalyzes the oxidation of a chromogen by the hydrogen peroxide to form a colored product. This method does not react with lactose, fructose or galactose. Study the dipstick color chart to become familiar with the range of color changes. The urine specimen should be at room temperature for these enzyme reactions to occur properly.

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False Negative Results

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.

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Test for Reducing Substances Other than Glucose

Urine specimens from certain pediatric patients should be tested for other reducing substances, such as galactose, when the results for glucose are negative using the routine dipstick method. The laboratory's procedure should define when additional testing is needed.

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Clinical Significance

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.

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Clinical Significance cont'd

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

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Three Kinds of Ketones

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.

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Testing for Ketone Bodies

Testing for ketone bodies is based on a nitroprusside reaction. Acetoacetic acid reacts with sodium nitroferricyanide and glycine in an alkaline medium to produce a violet-to-purple colored complex. The reagent strip method can detect as little as 5 mg/dL acetoacetic acid in urine. It does not react with acetone unless glycine is present or B-hydroxybutyric acid. Since these two compounds are derived from acetoacetic acid, their presence can be assumed if the test for ketones is positive. Ketones are reported either as negative, small, moderate or large amounts; or negative, 1+, 2+, 3+, or 4+. In some severe cases of ketosis, it may be necessary to perform tests on serial dilutions to provide more information on the quantity of ketones present.

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Ketone Bodies

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.

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False Positive Ketone Results

False positive ketone results may be seen in patients after BSP or PSP dye injection due to the phthaleins. The presence of L-DOPA metabolites, some urine preservatives (e.g. 8-hydroxyquinaline), or high levels of phenylketones will also cause false positive results. Antihypertensive drugs such as methyldopa and captopril also may produce false positive results.

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False Negative Results

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.

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Clinical Significance of Positive Urine Ketone Result

Ketone bodies are usually absent in urine. The presence of ketones in the urine probably indicates that the body is using fats rather than carbohydrates for energy. 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 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 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 of carbohydrates or increased loss of carbohydrates.

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Which of the following substances can cause a false positive result for ketones? (Choose ALL of the correct answers)View Page
Significantly increased levels of ketones are detected in the urine with which of the following conditions? (Choose ALL of the correct answers)View Page
Bilirubin Characterization

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.

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Test for Bilirubin

The test for bilirubin on the urine reagent strip is based on the formation of an azobilirubin compound resulting from a reaction of bilirubin in an acid medium with diazotized 2, 4 dichloroaniline. The color of this compound ranges through various shades of tan. Some sources describe the colors produced as shades of tan-to-pink-to-violet. Since other pigments in the urine may influence the test results, this test strip is more difficult to interpret than the others. Colors which are unlike either the positive or negative color blocks on the color chart may be due to the presence of bilirubin -derived bile pigments. Any urine which demonstrates an atypical color on the bilirubin test strip should be tested further. Even a slight change in color should be considered significant since bilirubin is never present in normal urine.

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False Positive Results

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.

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Confirmatory Testing

Confirmatory testing using an alternative method, such as Ictotest reagent tablets, can be performed when positive results are seen on the dipstick strip, when a red color forms on the strip, or when atypical color changes occur that are the result of bilirubin-derived bile pigments in the urine masking the bilirubin reaction.

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Clinical Significance

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.

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Which of the following may cause a false positive bilirubin result on a urine reagent strip?View Page
Which of the following may cause false negative bilirubin results on a urine reagent strip? (Choose ALL of the correct answers)View Page
Hematuria

The term hematuria is used to describe the presence of intact red cells in the urine. The urine may be cloudy/red or pink in color and red cells are visible upon microscopic examination. If the red cells have been destroyed, hemoglobin will be excreted in the urine. The term, hemoglobinuria, is used to describe this condition. The color of the urine will be pink or red but clear rather than cloudy. The presence of only five red blood cells per microliter of urine is considered to be clinically significant. For this reason, a chemical test is needed to detect quantities of blood too small to change the color of the urine. Microscopic examination is used to differentiate between hematuria and hemoglobinuria if the reagent test strip is positive for blood.

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The Test for Blood

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.

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False Positive Results

A false positive result for blood on the reagent strip can occur when oxidizing contaminants, such as hypochlorite (bleach), remain in collection bottles after cleaning. Contamination of the urine with provodine-iodine, a strong oxidizing agent, used in surgical procedures can result in a false positive reaction. Microbial peroxide found in association with urinary tract infections may also cause false-positive results. Capoten® (Captopril) can cause decreased reactivity. The muscle tissue form of hemoglobin, myoglobin is a well-known cause of false-positive reactions on the blood portion of the reagent strip. When tissue hemoglobin is present, the urine specimen has a clear red appearance. Patients suffering from muscle-wasting disorders or muscular destruction due to trauma, prolonged coma, or convulsions or individuals engaging in extensive exertion may have myoglobin in their urine. Specific tests for myoglobin, such as immunodiffusion techniques or protein electrophoresis, are needed to confirm the presence of this substance in a urine specimen. Levels of ascorbic acid normally found in urine do not interfere with this test.

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False Negative Results

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.

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Clinical Significance

No blood is found in the urine of healthy individuals although samples from menstruating females, frequently, but not always, test positive for blood. Hematuria is associated with renal or genital urinary disorders in which the bleeding is the result of irritation to the involved organs or trauma. Examples include renal calculi, pyelonephritis, glomerulonephritis, tumors, trauma or exposure to toxic chemicals or drugs and/or strenuous exercise. Hemoglobinuria may be due to the lysis of red cells within the urinary tract. If it is caused by intravascular hemolysis, the hemoglobin is then filtered through the glomeruli. In the normal individual, the hemoglobin molecule attaches to haptoglobin and in this way bypasses the kidney filtration system. When the hemoglobin/haptoglobin system is overwhelmed, as in cases of hemolytic anemia, severe burns, transfusion reaction, infection or strenuous exercise, hemoglobin passes into the urine.

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A urine sample is cloudy pink in appearance. The microscopic examination reveals the presence of intact red cells. The term used to describe these findings is:View Page
Which of the following substances cause a false positive result for blood on the urine reagent strip? (Choose ALL of the correct answers)View Page
Which of the following substances may cause a false negative result for blood on the urine reagent strip? (Choose ALL of the correct answers)View Page
Nitrite Test

The nitrites portion of the reagent strip provides a rapid screening test for the presence of gram-negative bacteria that are often responsible for urinary tract infections. Although urine cultures are still needed to confirm the diagnosis and monitor any urinary tract or kidney infection, the need for a culture may not be obvious because in some cases of early bladder infection, the symptoms may be vague or the patient may be asymptomatic. 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.

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Bladder Infections

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.

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Test Sensitivity

This test is sensitive to 0.06-0.1 mg/dL nitrite ion in urines with a low specific gravity and 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 because yeasts or gram-positive bacteria unable to reduce nitrites may be the causative agent.

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False Positive Results

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.

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False Negative Results

False negative results may occur in urine specimens that did not remain in the bladder a sufficient length of time for the bacteria to reduce a measurable quantity of nitrate to nitrite. Other reasons for false negative results include high specific gravity, ascorbic acid levels above 25mg/dL or low pH (<6). Less frequently, the cause may be due to a lack of sufficient nitrate in the diet (green vegetables) or further reduction of nitrite to nitrogen when large numbers of bacteria are present. In patients receiving antibiotics, the metabolism of the bacteria may be inhibited which would also produce a false negative reaction.

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Nitrates in urine are reduce to nitrites by:View Page
Match the following:View Page
Urobilinogen

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.

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False Positive Urobilinogen Results

A false positive urobilinogen reaction may occur with the dipstick method when substances known to react with Ehrlich's reagent such as sulfonamides and p-aminosalicylic acid are present in the urine. Drugs that contain Azo dyes, such as Azo Gantrisin®, have a gold color that masks the reaction, causing a false positive reaction. Atypical color reactions may be obtained in the presence of high concentrations of p-aminobenzoic acid. The dipstick urobilinogen test cannot detect porphobilinogen in a urine specimen. Porphobilinogen is a molecule formed during the synthesis of the heme portion of hemoglobin.

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False Negative Urobilinogen Result

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.

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Clinical Significance

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. Urobilinogen is increased in hemolytic disease and urine bilirubin is negative. Urobilinogen is increased in hepatic disease, and urine bilirubin may be positive or negative. Urobilinogen is low with biliary obstruction, and urine bilirubin is positive. Reagent strips methods however, cannot distinguish normal urobilinogen from absent urobilinogen, as might be seen in complete biliary obstruction.

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Which of the following cause false negative reactions when testing for urobilinogen with the reagent strips? (Choose ALL of the correct answers)View Page
Urobilinogen is excreted in the urine in increased amounts in: (Choose ALL of the correct answers)View Page
Granulocytic white blood cells

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.

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Leukocyte Esterase Dipstick Test

If leukocyte esterase is detected, 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; being aware that white blood cells may be absent if they are lysed, yet releasing their esterases into the specimen. Positive results may occasionally be found in random specimens from females due to contamination of the specimen by vaginal discharge.

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False Positive Leukocyte Esterase Test

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.

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A positive leukocyte esterase test indicates the presence in a urine specimen of which of the following?View Page
Measuring Specific Gravity

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.

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Advantages and Limitations of the Reagent Strip Method for Specific Gravity

Specific gravity (SG) measured with the reagent strip method correlates well with gravimetric measurement, and, unlike the gravimetric or refractometer methods, does not need to be corrected for glucose or protein. Cloudy/turbid urines do not need to be clarified before measuring SG with the reagent strip method. It is the recommended method for determining SG if a urine specimen contains x-ray contrast media or plasma expanders. Alkaline urine can affect the indicator system and lower the SG result on the reagent pad. If the result is being read visually, it is recommended that .005 be added to the SG result when the pH is alkaline. Most dipstick readers, however, will automatically adjust the SG reading for pH. A SG reading higher than the reagent strip range would need to be measured by another method, and may require dilution.

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A visually-read specific gravity (SG) on a urine specimen with an alkaline pH should be adjusted by:View Page
How does ion concentration in the urine relate to specific gravity (SG)?View Page
Semi-automated and automated reagent strip readers:View Page
Urine reagent strips readers use any manufacturers dipsticks.View Page
When an automated or semi-automated method is used to read urine reagent strips, quality control testing must be performed at least:View Page
Quality Control

Both a normal and an abnormal urine control must be tested with each new lot of reagent strips, and at least every day of patient testing to confirm the accuracy of the reagent strips and the dipstick reader. Some dipstick readers also require periodic calibration. Follow the manufacturer's instructions for calibration procedure and frequency. Quality control results must be recorded, and corrective action must be taken when the results are not in the acceptable range.

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Chemistry / Urinalysis Question Bank - Review Mode (no CE)
An increased number of these cells, when found upon microscopic examination of urine is termed:View Page
The cells present in this illustration are:View Page
The cells faintly seen between the arrows are consistent with:View Page
The cell indicated by the arrow is a:View Page
Identify the urine sediment elements shown by the arrow:View Page
Identify the urine sediment elements shown by the arrow:View Page
Identify the urine sediment elements shown by the arrow:View Page
Identify the urine sediment elements present in this acid urine:View Page
Identify the urine sediment elements shown by the arrow:View Page
Identify the urine sediment elements present in this illustration:View Page
Identify the urine sediment elements indicated by the arrow in the illustration:View Page
Which one of the following crystals is not found in normal urine:View Page
Identify the urine sediment elements indicated by the arrow in the illustration:View Page
Identify the sediment elements present in this alkaline urine:View Page
Identify the urine sediment element indicated by the arrow in the illustration:View Page
Identify the urine sediment elements shown by the arrow:View Page
Identify the urine sediment elements present in this illustration:View Page
Identify the urine sediment elements shown by the arrow:View Page
Identify the urine sediment elements shown by the arrow:View Page
Identify the urine sediment elements shown by the arrow:View Page
Which two of the following crystalline elements are found in acid urine:View Page
Identify the urine sediment element shown by the arrow:View Page
Identify the urine sediment elements shown by the arrow:View Page
Identify the urine sediment elements shown by the arrow:View Page
Identify the urine sediment elements shown by the arrow:View Page
Identify the urine sediment elements shown by the arrow:View Page
Identify the urine sediment element shown by the arrow:View Page
Which of the following methods would be used to confirm the presence of Bence-Jones protein in the urine:View Page
Which two of the following test combinations could best be used to help rule out an ectopic pregnancy:View Page
A urine specimen which was collected in a dark container, stored in the dark, has a pH of 7.5, and whose sample aliquot is wrapped in foil, is most likely being sent for:View Page
Which of the following tests would be used in the assessment of glomerular filtration:View Page
Match urine color with substance that might have been responsible:View Page
Which of the following is not a standard method for measuring the specific gravity of urine:View Page
The normal range for urine pH is:View Page
The term specific gravity is most closely related to which of the following:View Page
Which of the following cells when found upon microscopic examination of the urine would be most indicative of kidney disease:View Page
Which of the following casts might be found in urine of a healthy individual after strenuous exercise:View Page
Which two of these urine elements are found exclusively in acid urine:View Page
Bence-Jones proteinuria can be seen in all of the following conditions except:View Page
The renal threshold is best described as:View Page
Bacterial contamination of a urine specimen from a normal healthy individual could originate from all of the following except:View Page
Detection of a fruity odor in a fresh urine sample may be indicative of:View Page
In a patient with acute glomerulonephritis you would expect to find all but the following in the urine except:View Page
Which of the following methods may be employed to definitively identify Bence-Jones proteins:View Page
An average adult would excrete approximately what volume of urine per 24 hours:View Page
Which of the following would be the most appropriate method to confirm a positive protein from a urine dipstick:View Page

Confirmatory and Secondary Urinalysis Screening Tests
Diseases Associated with Proteinuria

Normal urine contains very little protein, usually less than 10 mg/dL. The majority of the protein that is found in normal urine is albumin. The presence of an increased amount of protein in the urine (proteinuria) can be an indicator of renal disease. The two mechanisms that can lead to proteinuria are glomerular damage or a defect in the reabsorption process of the tubules in the nephron. The concentration of protein in the urine is not necessarily indicative of the severity of renal disease. Severe proteinuria (greater than 3.5 g/day) is characteristically seen in patients with glomerulonephritis, lupus nephritis, lipoid nephrosis, and severe venous congestion of the kidney. Moderate proteinuria (0.5-3.5g/day) is seen in nephrosclerosis, multiple myeloma, diabetes nephropathy, malignant hypertension, and pyelonephritis with hypertension. Mild proteinuria (less than 0.5 g/day) may be seen with polycystic kidneys, chronic pyelonephritis, benign orthostatic proteinuria, and some renal tubular diseases. Transient proteinuria can also be due to physiologic conditions such as stress, exercise, cold exposure, and fever, in the absence of renal disease.

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Screening and Secondary Tests for Protein

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.

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Sulfosalicylic Acid Test (Exton's Modification)

There are several acids which can be used to precipitate proteins - sulfosalicylic, trichloroacetic, nitric, and acetic acids. Sulfosalicylic acid (SSA) is the most frequently used acid test because it does not require the use of heat. Exton's reagent is 5% sulfosalicylic acid in a solution of sodium sulfate. Exton (1925) found that adding sodium sulfate to the SSA causes a more uniform precipitate to be formed. To perform the SSA procedure mix equal parts of patient urine and the reagent. Rate the amount of turbidity according to the following scale:

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Microalbumin Test

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.

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SSA Procedural Notes and Test Limitations

When testing for protein using the SSA precipitation method, a urine sample that is not clear should be centrifuged and the supernatant used for testing.Daylight or fluorescent light is recommended when interpreting results. Quality control checks should be performed by testing known negative and positive specimens or controls. False-positive results can occur when the urine specimen:Contains x-ray contrast mediaContains a high concentration of certain antibioticsIs turbidFalse-negative results can occur when the urine specimen:Is a highly buffered alkaline urine (may need to acidify the urine to a pH of 7.0 or lower before performing the SSA test).Is very dilute Is turbid

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Persons with type 1 or type 2 diabetes should periodically have their urine monitored for which of the following to avert renal complications related to diabetes?View Page
The screening test most commonly used to detect Bence-Jones protein in urine is:View Page
Which of the following conditions may require the use of the sulfosalicylic acid (SSA) test to detect protein as part of a routine urinalysis?View Page
Albumin is the main serum protein found in normal urine.View Page
Heat and Acid Test for Urinary Protein

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Urine Bilirubin

Bilirubin is a degradation product of hemoglobin. When red blood cells (RBCs) have reached the end of their normal life span (approximately 120 days), they are destroyed in the spleen and liver. Hemoglobin that is freed in the process is further broken down into iron, protein, and protoporphyrin. Protoporphyrin is converted to bilirubin and released into the circulation. Bilirubin binds to albumin and is transported in the blood to the liver. This unconjugated bilirubin is insoluble in water and cannot be filtered through the glomerulus of the kidney. Bilirubin is then conjugated with glucuronic acid in the liver. This conjugated bilirubin is water soluble and is excreted by the liver through the bile ducts and into the duodenum; bilirubin does not normally appear in the urine. However, if the normal degradation cycle is disrupted, as happens with cirrhosis, hepatitis, and other conditions that damage the liver, conjugated bilirubin will appear in the urine. Since conjugated bilirubin is not bound to protein, it is easily filtered through the glomerulus and excreted in the urine whenever the plasma bilirubin level is increased.

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A urine specimen is received in the laboratory late in the afternoon. The specimen was collected early in the morning and was accidentally left in bright sunlight and at room temperature on a counter in the outpatient clinic. The test order is for urine bilirubin screening. Which of the following could occur as a result of the storage conditions?View Page
Ictotest® Procedure

Supplies needed to perform the Ictotest® include the Ictotest® reagent pad, reagent tablet, water and the urine specimen.

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Procedure

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.

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Urine Bilirubin

Normally, small amounts of conjugated bilirubin, regurgitate back from the bile duct and enter the blood stream, so small amounts of conjugated bilirubin can be found in the plasma. Since conjugated bilirubin is not bound to protein, it is easily filtered through the glomerulus and excreted in the urine whenever the plasma level is increased. Normally, no detectable amount of bilirubin (sometimes referred to as "bile") is found in the urine.

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Bilirubinuria

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).

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Confirmation of Urine Bilirubin Result

The reagent strip test for bilirubin may not be sensitive enough to detect small amounts of bilirubin in urine, which may be present in the earliest phases of liver disease or viral hepatitis. The Ictotest® reagent tablet is more sensitive and is recommended when bilirubin in the urine is particularly of interest. False-positive results can occur in screening procedures for bilirubin due to color interference from large amounts of blood in the urine, very concentrated urine, or drugs that discolor the urine, such as phenazopyridine (Pyridium). Because of this, it is important to verify positive or questionable bilirubin results with a confirmatory method, such as the diazo tablet test, available commercially as the Ictotest®. 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.

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Reporting Ictotest® Results

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.

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Limitations of the Procedure

The product profile for Ictotest® points out that bilirubin is very light sensitive, so urine specimens should be protected from excessive light exposure and examined as soon as possible after specimen collection. On standing, bilirubin, which has a goldish color, is oxidized to biliverdin, which is a green color. Many of the procedures used to detect bilirubin will not react with biliverdin, so false-negative results may occur if urine is not fresh when tested.

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Reasons for Performing Confirmatory or Secondary Macroscopic Urine Tests

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.

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Urine Glucose

The presence of significant amounts of glucose in the urine is called glycosuria (or glucosuria). The amount of glucose present in urine is dependent upon the blood glucose level, the rate of glomerular filtration, and the degree of tubular reabsorption of the sugar. Usually glucose will not be present in the urine until the blood level exceeds 160-189 mg/dL, which is the normal renal threshold for glucose. The main reason for glycosuria is an elevated blood glucose level (hyperglycemia). Diabetes mellitus is the most common cause of hyperglycemia. However, stress, obesity, brain injury, myocardial infarction, hyperthyroidism, pregnancy, and a lowered renal threshold due to kidney damage can all cause glycosuria.

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Reducing Sugars

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.

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Specimen Processing for Urine Sugar Testing

Prompt testing (within two hours of urine specimen collection) or refrigeration of the specimen is necessary because the glycolytic enzymes from the cells and bacteria, if present, will decrease the sugar in the urine.

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The Clinitest® Method

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.

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Testing for Reducing Substances Other Than Glucose

Some laboratories perform testing for other reducing substances on urine specimens from children below a certain age (eg, age 2) whenever a urinalysis is ordered. The purpose is to detect serious inborn errors of metabolism. The reducing substance assay uses the classic Benedict’s copper reduction reaction to detect glucose and other carbohydrate metabolites. If the reducing substance test (ie, Clinitest®) is positive and the reagent dipstick assay for urinary glucose is negative, it is possible that other reducing substances are present. Further testing would be required to diagnose the exact inborn error of metabolism. With the current practice in most U.S. states of screening all newborns for metabolic disorders, the urine test for reducing substances may be performed only when the clinician orders it, rather than the test being part of a routine urinalysis procedure. Clinitest® is not the recommended monitoring test for diabetics; glucose reagent strip testing is a better choice.

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Testing Methods for Urine Glucose and Other Reducing Substances

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.

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The 2-Drop Clinitest® Procedure

Place 2 drops of urine in a test tube.

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Causes of Ketonuria

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.

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Detection of Ketone Bodies

The terms "ketones" or "ketone bodies" refer to acetoacetic acid (acetoacetate), acetone, and beta-hydroxybutyric acid (BHB). Screening procedures used to detect ketonuria do not react with all ketones. Acetoacetate reacts most often in the reagent strip screening test methods. Acetoacetate and acetone react in the Acetest® method.

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Correlation of Urine Glucose and Ketones

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.

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Urinary Ketones

Ketone bodies are formed in the liver as intermediates in the catabolism of fatty acids. In healthy individuals, ketone bodies are almost completely metabolized so that only negligible amounts appear in the urine.

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Acetest®

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.

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Department of Transportation (DOT) Federally Regulated Urine Specimen Collection Training
Observed collection

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.

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Federal drug testing custody and control form (CCF)

The federal drug testing custody and control form (CCF) must be used to document every urine collection required by the Department of Transportation drug testing program. At the present time, these include the: Federal Motor Carrier Safety Administration (FMCSA) Federal Aviation Administration (FAA) Research and Special Programs Administration (Pipeline) (RSPA) Federal Transit Administration (FTA) Federal Railroad Administration (FRA) United States Coast Guard (USCG)

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Non-federally regulated custody and control form

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.

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Intent of this program

This program is intended to provide guidance and training to those individuals who will be conducting both Department of Transportation (DOT) and Department of Health and Human Resources (HHS) regulated urine specimen collections. While this program is more than just an overview, obvious restraints prohibit an in-depth discussion of every procedure or problem that might be encountered. This program only serves as a training program. It does not represent final authority. Every effort has been taken to keep this course up-to-date with current regulations. However, if anything in this program conflicts with the Urine Specimen Collection Guidelines, U.S. Dept. of Transportation, Office of Drug and Alcohol Policy and Compliance (Aug. 31, 2009), that reference prevails and must be followed.Training to qualify as a drug screen collector must include the flawless completion of five mock collections. These mock collections must include the following scenarios and must be performed in the presence of a qualified collector: Two uneventful collections. One collection in which the quantity of specimen is not sufficient. One collection in which the temperature of the specimen is out of range. One collection in which the donor refuses to sign either the donor certification on the Medical Review Officer's (pink) copy of the CCF or refuses to initial the security strips.

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Five areas having prerequisites for proper collection

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.

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Other collection site requirements

In addition to the security concerns listed in the previous slide, a collection site must have: A restroom for the donor to have privacy while providing the urine specimen. A single commode restroom with a full-length privacy door is preferred. However, a multi-stall restroom with a partial-length door is acceptable. Both facilities should be large enough to accommodate two individuals in the event of an observed collection. A source of water for hand washing. It is preferred that it be external to the restroom where urination occurs. A suitable clean surface for the collector to use as a work area and for completing the required paperwork.

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Collection site security requirements

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.)

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Collection Supplies

As collector:You can process only ONE donor at a time. You may not act as the collector for anyone whom you immediately supervise unless no other qualified collector is immediately available. You can not collect your own urine specimen. You should have appropriate identification available should the donor request it. This identification is limited to your name and the collection company where you work. You are not required to show documentation of training unless requested by a DOT representative, state government representative, or an employer. You should keep a file of the names and telephone numbers of Designated Employer Representatives (DER) to contact about any problems or issues that may arise during the collection process.

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Acceptable forms of identification

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.

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The collection kit

Let the donor select a collection kit. There should be 10 or more kits available from which the donor may chose. The kit is comprised of a collection cup, leak resistant plastic bag, and two specimen vials. You or the donor may open the collection kit in the other's presence. The donor is given the collection cup. It is the only item that can be taken into the restroom. The specimen vials remain with the collector. Do not open these vials until you are ready to disperse the urine specimen.

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Collector disperses specimen to bottle(s)

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.

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Collector applies tamper-evidence seals

After dispensing the urine specimen into the specimen vials, the collector, not the donor, removes the tamper-evidence seals from the control form and places them on the specimen vials. Seal "A" goes over the primary vial containing 30 mL; seal "B" goes over the secondary vial containing 15 mL. (When doing a Non-Regulated drug screen, since only one vial would be used, "A" would be the appropriate tamper-evidence seal to use.)The seal must be centered over the lid and down the sides of the vial to ensure that the lid cannot be removed without destroying the seal.

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Steps for Typical Urine Collection

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.

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Refusal to test

When a donor behaves in a confrontational way that disrupts the collection process, such as refusing to empty pockets or refusing to wash hands after being directed to do so by the collector, this is considered interfering with the testing process and is considered a refusal to test. The collector must stop the collection then contact the Designated Employer Representative (DER) regarding the refusal either by telephone, email, or secure fax to ensure that notification is immediately received. The collector must document the refusal to test on the Federal Custody and Control Form (CCF) and send all copies of the CCF to the DER.If a donor makes an attempt to provide a specimen and the quantity is not sufficient (QNS) and the donor refuses to make a second attempt to provide another urine specimen or leaves the collection site before the collection process is completed, this is considered a refusal to test. The collector must stop the collection and discard any urine collected. The collector must notify the DER as outlined above, document the refusal to test on the Federal CCF and forward all copies of the CCF to the DER.

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Situations not considered as refusal

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.

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Shy bladder

The term "shy bladder" refers to a situation where the donor is unable to provide the sufficient amount of urine required for a drug screen.If the donor indicates upon arrival at the collection site that he or she cannot provide a specimen, the collector should begin the collection process anyway and have the donor make an attempt to provide a specimen. If after an attempt the donor cannot provide a specimen or can only provide a specimen of insufficient volume, the donor must be instructed not to leave the collection site and to do so will be considered a refusal to test. The donor should be monitored either by the collector or by another member of the collection site staff. The donor should be encouraged to drink up to 40 ounces of fluid reasonably distributed over a period of up to three (3) hours, or until the donor can provide a sufficient amount of urine, which ever comes first. If no specimen is provided on the first attempt, the same collection container may be used for the next attempt. The donor may keep possession of the container during the waiting period. The same CCF is used.

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Shy bladder

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.

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When problems occur

Fortunately, the great majority of collections are uneventful, but from time to time problems or the unexpected occur. This section will discuss a few examples of special situations that may take place during a collection and what the response of the collector should be. Obviously, not every special situation can be envisioned or discussed. It is strongly recommended that the collector be very familiar with the Department of Transportation publication: Urine Specimen Collection Guidelines dated October 1, 2010.

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Observed collection scenarios

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.

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Fatal Flaws and Correctable Flaws

Fatal FlawsIt is important to remember that the following are fatal flaws and can cause the specimen not to be tested: Number on Custody and Control Form and security strips do not match. Security strip on the specimen vial is broken or shows evidence of tampering. Quantity of urine needed is not sufficient. There is no printed collector's name or signature.Correctable FlawsThe following are flaws that may be corrected by either sending a signed statement or a Memorandum for Record to the laboratory: The collector printed his or her name, but forgot to sign the CCF. The collector checked the temperature of the specimen, but forgot to note this fact on the CCF.

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Responsibilities and requirements for collectors

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.

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Diabetes and the Current American Diabetes Association Guidelines
Other Complications

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.

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Clinical Testing

A large number of assays related to carbohydrate management and diabetes monitoring are performed in clinical laboratories, hospital nursing units, nursing homes, physician offices, clinics, and by patients at home, school, or work.Assays that will be discussed are: Blood Glucose Urine Glucose Ketones Microalbuminuria Insulin and C-Peptide Insulin Antibodies Glycosylated Proteins

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Urine Glucose

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.

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Urinary Albumin Excretion

Screening for early occurrence and low amounts of albumin in urine detects microvascular disease before impaired renal function and insufficiency occur. Regular screening of urinary albumin excretion (UAE) is recommended for individuals with both type 1 diabetes and type 2 diabetes as an early indicator of renal disease. It is recommended at the time of initial diagnosis and annually thereafter for patients with type 2 diabetes, and commencing annually 5 years after the initial diagnosis of type 1 diabetes. Control of blood pressure and blood glucose concentrations can slow the rate of renal function decline.

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Microalbuminuria

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.

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Ketones

Acetyl CoA is converted to acetone, acetoacetate, and beta-hydroxybutyrate. These are acids and when dissolved in body fluids in excess lower the blood pH. Increased ketones can result in a metabolic acidosis referred to as ketosis, ketoacidosis or diabetic acidosis. Type 1 diabetic patients are especially at risk for ketoacidosis. Urine and serum ketones are measured semiquantitatively and a diabetic in ketosis is monitored for ketones and blood pH.

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Electrophoresis
Specimens

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.

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After reviewing the information on specimen samples for electrophoresis, select the one correct statement.View Page
Routine 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.

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High Resolution Electrophoresis (HRE)

High resolution electrophoresis (HRE) is routine electrophoresis using a high voltage. Serum samples separated with HRE may yield approximately fifteen distinct protein bands. Other HRE applications are the separation of CSF proteins for the diagnosis of multiple sclerosis and light chains in urine for early detection of lymphoproliferative disorders such as multiple myeloma. Both of these specimen separations require more resolution of proteins than routine protein electrophoresis can provide. Increasing the voltage will increase heat generated. To prevent denaturation of proteins, drying out of gels and other system components, a cooling system is included in HRE instrumentation.

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Electroendosmosis

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.

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Resurgence of Electrophoresis

Traditionally, most clinical laboratory electrophoresis utilizes methods that separate and identify proteins in serum, urine, CSF, and some other body fluids. Most studies are designed to detect serum protein abnormalities and gathering more information about gammopathies.In recent years, there has been a resurgence in electrophoresis use and methods. Development of automated methods has enhanced this. The evolution of numerous molecular diagnostic investigations and research in proteomics have also augmented electrophoresis.Applications of two-dimensional electrophoresis discussed the use of electrophoresis in proteomics. Electrophoresis and molecular diagnostics, blotting techniques, and current uses of CE in molecular diagnostics will be discussed now.

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General Laboratory Question Bank - Review Mode (no CE)
What is the primary functional unit of the kidney :View Page
Which of the following tests would be useful in the assessment of glomerular filtration:View Page
Match urine color with substance that might have been responsible:View Page
Which of the following is not a standard method for measuring the specific gravity of urine:View Page
The normal range for urine pH is:View Page
Which of the following cells when found upon microscopic examination of the urine would be most indicative of kidney disease:View Page
Which of the following casts might be found in the urine of a healthy individual after strenuous exercise:View Page
Match the urine sediment or crystal to the correct description.View Page

Hereditary Hemochromatosis (retired 2/13/2014)
Iron Intake and Recycling

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)

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HIV Safety for Florida
Which of the following is not considered a potentially infectious body fluid for transmitting HIV?View Page
Which of the following does not pose a significant risk for transmitting HIV?View Page

Introduction to Bioterrorism
Laboratory Response - Chemical, Level 2

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.

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Laboratory Response - Chemical, Level 1

At present, 5 laboratories participate in Level 1 activities. At this level, technical personnel are trained to detect exposure to an expanded number of chemicals in human blood and urine. This includes all Level 3 and 2 laboratory analyses, plus analyses for mustard agents, nerve agents, and other toxic chemicals.

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Laws and Rules of the Florida Board of Clinical Laboratory Personnel (retired 9/1/2010)
Description of Specialties (2)

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.

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Description of Specialties (3)

Specialists in radioassay use radionuclides to determine the chemical makeup of body fluids such as blood and urine. Specialists in blood gas analysis evaluate lung and breathing function by levels of oxygen, carbon dioxide, pH, and hemoglobin with automated tests. Specialists in histology examine cellular and tissue samples using fixation, dehydration, embedding, microtomy, frozen sectioning, staining, and other similar techniques. Histology specialists licensed as technicians can perform specimen processing, embedding, cutting, staining, and frozen sectioning only under the general supervision of a director, supervisor, or technologist. Specialists in cytology process and interpret samples relating cytopathological disease. Non-gynecological cytology preparations can be screen by a specialist in cytology but final review and interpretation must be done by a physician.

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Medicare Compliance for Clinical Laboratories (retired)
Confidentiality and inducements

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.

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Microbiology / Serology Question Bank - Review Mode (no CE)
This suspicious form, found in urine, measures 120 micro meters by 50 micro meters.View Page
When a urine specimen cannot be cultured immediately it may be:View Page
Which of the following specimens is the most sensitive for detecting active CMV infection:View Page

Mycology: Yeasts and Dimorphic Pathogens (retired 2/12/2013)
Match the name of each species of yeast listed below with the location listed in the drop-down box where that species may be concentrated.View Page
Match the complications that are most likely to be associated with each of the two yeast diseases that are listed in the drop-down box:View Page
Illustrated in this photomicrograph of a lactophenol blue preparation of a urine sediment is a cluster of yeast cells that were presumptively identified as Cryptococcus species. Further characteristics that may assist in confirming this identification are:View Page
This photomicrograph is a representative field of a Wright-Giemsa-stained bone marrow aspirate in which a pair of budding yeast cells is seen centrally (arrows). Based on the appearance of these yeast cells, what other test would you expect to be positive?View Page

Parasitology Question Bank - Review Mode (no CE)
Which of the following specimen types is/are suitable for the recovery of Entamoeba histolytica?View Page
The specimen of choice for the recovery of Entamoeba hartmanni is:View Page
This suspicious form, found in urine, measures 120 µm by 50 µm.View Page
Match each parasite listed here with its corresponding optimal specimen type from which it may be recovered: (Answers may be used more than once.)View Page
Stool is the specimen of choice for the recovery of which of the following parasites?View Page

Pharmacology in the Clinical Lab: Therapeutic Drug Monitoring and Pharmacogenomics (retired 10/15/2012)
Polymorphism and CYP450

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.

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Genotype versus Phenotype

Phenotyping involves measuring the metabolism of a probe drug. For example, with CYP2D6, dextromethorphan or debrisoquine can be given to a patient to see how well the drug is metabolized. Both these drugs are safe and extensively metabolized by CYP2D6. By measuring the parent drug and the metabolite in urine, the metabolic capacity of a CYP450 enzyme can be estimated. Such testing is complex and tedious, however, and has not become routine in clinical laboratories. Therefore, genotyping is likely to be the main tool that is used for assessing the PGx of a patient.

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Phlebotomy
What is a phlebotomist? [continued]

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.

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Specimen collection

To screen for gestational diabetes, collect blood after one hour.For a standard glucose tolerance test collect blood and urine at 30 minute intervals, for two hours.To confirm gestational diabetes, collect blood every hour for 3 hours.

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Urine

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.

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Collection kits

Sealed collection kits are opened in the presence of the donor individual.The kit contains detailed directions and materials for urine and blood collection. Use only the materials supplied in the kit.You may have to appear in court later to testify as to how you collected the specimens, and to verify their origin, so follow directions carefully.

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Urine continued

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.

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Reading and Reporting Gram Stained Direct Smears
Determine the Quality of a Urine Specimen Submitted for Culture

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."

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Size and Appearance of Nonbacterial Cellular Elements on Gram Stained Smears

Type of Cell Average Size Image Comments Epithelial cells 25 µm Appear pink/red on Gram stained smear. Larger than white blood cells. Have a single nucleus. They are an indication of a suboptimal or unacceptable specimen if present in large numbers in sputum specimens, tracheal or endotracheal aspirates, or in urine specimens. White blood cells 12 µm Appear pink/red on Gram stained smear. Most often, polymorphonuclear white blood cells (PMNs). White blood cells indicate inflammation and possible infection. The direct smear examination should focus within and around these cells. Hyphae/pseudohyphae Varies Appear blue on Gram stained smear. Hyphae are tubular filamentous fungal elements, which may show branching or intertwining. Pseudohyphae are multiple buds of yeast that do not detach, thereby forming chains. Yeast 7 µm Appear blue on Gram stained smear. Round to oval, often budding. About the same size as red blood cells. Generally much larger than bacteria. A few 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. Red blood cells 7µm Appear red on Gram stained smear Not usually considered a significant finding.

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Reading Gram Stained Direct Smears
Significance of Specific Findings:

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.

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Risk Management in the Clinical Laboratory
Preanalytic Phase

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.

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Special Topics in Phlebotomy
Screening for Diabetes Mellitus and Gestational Diabetes

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.

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The Urine Microscopic: Microscopic Analysis of Urine Sediment
Accurate microscopic results can be obtained on a urine specimen that has been refrigerated for up to:View Page
The volume of urine recommended for centrifugation for a microscopic examination is:View Page
Common crystals which can be found in ACID urine include: (Choose all that apply.)View Page
Abnormal crystals that can be found in urine include: (Choose all that apply.)View Page
Use the following urinalysis report to answer:The patient is a female and the urinalysis is completed within two hours of collection.Color - light yellow Appearance - slightly turbid Sp. Gravity - 1.009 pH - 8.0 Glucose - negative Protein - 1+ Blood - negative WBC - 5/HPF RBC - 1/HPF Epithelial - 0/HPF Casts - 2 hyaline/LPF Crystals - amorphous urates Bacteria - 2+True or false? The results are abnormal but all results correlate.View Page
Which of the following findings may correlate with the presence of a yeast infection?View Page
Formation and Significance of Casts

Casts are cylindrical bodies formed either in the distal convoluted tubules or the collecting ducts of the kidney. Since the walls of the tubule act as a mold for cast formation, the width of the tubule determines the width of the cast. Thus, narrow casts are formed in the distal tubules while broad casts are formed in the collecting ducts. The matrix of all casts is thought to be Tamm-Horsfall protein, a glycoprotein secreted by the distal loop of Henle and the distal tubule. This protein entraps cells and granular material of tubular origin. Very few casts are seen in the urine of a person without renal disease, except for hyaline casts, which may be transiently present after strenuous exercise, and during fever, diuretic therapy, and congestive heart failure. A significant number of urinary casts usually indicates the presence of renal disease.

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Which of the following are characteristics of casts? (Choose all that apply.)View Page
All of the following factors favor cast formation EXCEPT:View Page
Factors Promoting Cast Formation

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.

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Hyaline Casts

Hyaline casts are the type most commonly seen in the urine sediment. A few hyaline casts may occasionally be found in normal urine, and hyaline casts may be seen after strenuous exercise, during fever, or when undergoing diuretic therapy. Pathologically, hyaline casts may be seen with congestive heart failure, and may be seen together with other types of casts in a variety of renal diseases. Hyaline casts have a refractive index similar to the urine in which they are suspended. For this reason, hyaline casts will appear almost invisible under brightfield microscopy, but are easily seen with the use of phase-contrast microscopy.

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The Urine Microscopic Exam

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.

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The formed elements which may be present in urine sediment include: (Choose all that apply.)View Page
Microscopic Examination

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.

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Urine Specimen Collection

A urine specimen for urinalysis should be collected in a clean, dry, disposable container. If the sample is to be cultured, the container must be sterile. The preferred method is the " midstream clean catch" collection. The external genitalia are cleansed with a mild antiseptic solution. The first part of the urine stream is discarded while collecting only the midstream portion of the urine.

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Specimen Collection and Storage

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.

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Microscopic Examination of Urine Sediment

The sediment may be examined using both brightfield and phase-contrast microscopy. With the brightfield microscope, subdued light must be used. Some structures will be missed if there is too much light in the field. Fine focus throughout the examination to identify structures in different focal planes. Scan the slide on low power for quantification of casts, crystals and elements that are present in only a few fields. Use high power to identify casts and count red blood cells, white blood cells and epithelial cells.

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The volume of urine that is centrifuged and the amount of sediment that is used for microscopic examination should be the same for all persons performing urine microscopic examinations within a given laboratory.View Page
Steps in Preparing a Concentrated Urine Sediment

If a glass slide and coverslip are used to exam the urine sediment, these procedures should be followed:Mix urine specimen well, and transfer a standardized volume into a conical centrifuge tube. Use the volume that is stated in your laboratory's procedure. Centrifuge at the speed and spin time specified in your laboratory's procedure. Decant the sample to a standard volume (e.g., 0.5 or 1.0 mL) and resuspend the sediment in this volume. Pipette a specified amount of sediment onto the slide and put the coverslip in place.Ensure that the specimen fills the area under the coverslip without overflowing it. Ensure that there are no bubbles in the specimen under the coverslip. Upon examination, if cellular elements are not evenly distributed, a new suspension should be prepared.Microscope slides that are manufactured specifically for microscopic examination of sediment samples are available from several manufacturers. The slides are divided into standardized chambers to regulate the amount of urine sediment that is loaded for viewing These methods reduce reporting differences among testing personnel by ensuring that the same volume of sediment is consistently viewed and the sediment is viewed in a single plane.

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Cuboidal Cells

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.

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Urine Sediment

Urine sediment may also contain white blood cells (WBCs). Most of the WBCs in urine are segmented neutrophils. Since it is possible that lymphocytes, monocytes, and/or eosinophils may be present, the cells in urine can be stained if it is necessary to differentiate them. The segmented neutrophil indicated by the blue arrow shows a distinct nucleus. When viewing urinary sediment under the microscope, the fine focus adjustment must be used to identify white blood cells. White blood cells swell in dilute alkaline urine and the cytoplasmic granules exhibit brownian movement resulting in "glitter cells." These cells lyse rapidly. "Glitter cells" are most easily seen when viewed under phase-contrast microscopy.

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Swollen RBCs

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

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Characteristics of Cellular Elements

To review the characteristics of the cellular elements that can be seen in urine, study the following table.CellSignificanceEpithelial - SquamousMust be differentiated from other cells.Epithelial - Cuboidal or Renal TubularIncreased numbers indicate tubular necrosis, particularly important in renal graft rejection. Tubular damage caused by viral or bacterial infections.Epithelial - Transitional or CaudateNot significant unless found in large numbers or abnormal in appearance.White Blood Cells (WBCs)Bacterial infection.Red Blood Cells (RBCs)More than an occasional RBC can be significant. They are often associated with damage to the glomerular membrane of vascular injury within the genitourinary tract.Ghost Cells Alkaline urine causes RBCs to lyse, their empty membranes are called "ghost cells." Empty RBCs have the same significance as RBCs.YeastCan be confused with RBCs unless they are budding. Yeast can be seen in diabetes mellitus or in patients with vaginal moniliasis.BacteriaMay be a contaminant unless WBCs are present.

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Squamous Epithelial Cells

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.

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Large Clumps of White Cells

Large clumps of white cells, such as the ones shown in the slide, are typically found in chronic infection. The clumping is due to increased mucus in the urine.

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All of the following are characteristic of WBCs under high power (400X magnification) brightfield microscopy EXCEPT that they:View Page
Crenated Red Cells

This view shows crenated red cells which are found in concentrated urine. They appear as small cells with crinkly edges.

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Cell Types Observed in Urine Sediment

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.

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Renal Tubular Epithelial Cell

Another type of epithelial cell is the renal tubular epithelial cell. The proximal and distal convoluted tubules are the sites of origin for one form of these cells. They occur singly and are large (14-60 microns). Papanicolaou stain is useful in distinguishing renal tubular cells from other mononuclear cells in urine. Increased numbers of proximal and distal convoluted renal epithelial cells are seen in cases of acute tubular necrosis and certain drug or heavy metal intoxication.

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Red Blood Cells

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In concentrated urine, red blood cells (RBCs) may appear:View Page
Tyrosine Crystals

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.

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Summary of Abnormal Crystals

The characteristics of the more common types of abnormal crystals are summarized in the table below. CrystalColorSignificanceLeucineYellowMetabolismTyrosineColorless–yellowLiver disease (rare)CystineColorlessCystine metabolismCholesterolColorlessRenal tubular diseaseBilirubinGold-orangeIncreased bilirubinHigh doses of ampicillin, sulfonamide drugs, or other drugs may also result in urine crystal formation. It is important to check the patient's current medications when unusual crystals are found in the urine specimen.

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Cholesterol Crystals

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.

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Cystine Crystals

Cystine crystals indicate an abnormality in metabolism of the amino acid cystine. These crystals appear as colorless, refractile, hexagonal plates with even sides. Cystine crystals sometimes occur in pairs. They produce a red color in the nitroprusside reaction. Cystine may be confused with the hexagonal forms of uric acid but does not polarize light. Cystine crystals occur in acid urine.

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Bilirubin Crystals

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.

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Specimen #3 - Adult Female

The results are abnormal. The presence of glucose is not a normal finding. However, the two glucose methods correlate well with each other.The specific gravity does not correlate well with the glucose. A large amount of glucose should elevate the urine specific gravity. The specific gravity result should therefore be rechecked before reporting. The presence of 3+ bacteria, does not correlate well with scant white cells and lack of turbidity. The technologist should question whether the specimen was held at room temperature for a protracted period prior to examination.

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Crystals

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.

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Amorphous Urates

Amorphous urates appear as dark or yellow red granules while phosphates are white or colorless. The pH of the urine determines the type of amorphous crystals present. They may be urates in acid urine or phosphates in alkaline urine.

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Review of Common Crystals

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

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Identification of Crystals

Identification of crystals found in the urine sediment requires knowledge of the urinary pH. Large crystals are identifiable under low power. High power magnification is required for smaller crystals. Most crystals can be identified by morphology alone. Urine pH and reagent strip results can provide supporting information. If further examination is necessary birefringence and solubility characteristics should be performed.

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Crystals in Normal Acid Urine

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

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Crystals in Normal Alkaline Urine

Crystals found in normal alkaline urine include triple phosphate, ammonium biurate, calcium carbonate, and amorphous phosphates. This slide shows an example of triple phosphate crystals. These may appear as four to six sided prisms resembling coffin lids. They indicate either stasis of the bladder or a stale sample.

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Ammonium Biurate 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.

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Which of the following may be found in normal ALKALINE urine? (Choose all that apply.)View Page
Please identify these crystals occurring in ACID urine.View Page
Which of the following may be found in normal ACID urine?(Choose all that apply.)View Page
An Introduction to Quantitating the Urine Microscopic

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

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Introduction

In previous exercises we have examined the formed elements of the urine sediment including casts, cells, crystals, and miscellaneous structures. If the urine sediment contains only a few elements, identification may be simple. However, a sediment may contain an overwhelming number of elements. If this is the case, there are biochemical tests to aid in differentiation of structures.

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A urine specimen was delayed in transport to a medical laboratory. Upon examination of the urine sediment, the technologist cannot distinguish between bacteria and amorphous urates. Which of the following reagents would dissolve the crystals?View Page
A technologist is trying to enumerate the number of fat droplets in a urine sediment which also contains numerous red blood cells. Which of the following reagents would stain the fat droplets orange red?View Page
Staining Eosinophils

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.

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Parasites

Parasites which may be found in urinary sediments include Trichomonas vaginalis, Enterobius vermicularis and Schistosoma haematobium. It is also important to note that parasites and parasitic ova may be seen in urine sediments as a result of fecal or vaginal contamination. This slide shows examples of Trichomonas vaginalis. In the female, Trichomonas is usually found as a contaminant from vaginal infection and is often accompanied by an increase in the number of white cells. Trichomonas is highly motile, measuring 5 - 15 microns with a characteristic pear shape. It has multiple anterior flagella and the nucleus is often apparent.

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Enterobius Vermicularis (Pinworm) Ova

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.

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Oil or Fat Droplets

Oil or fat droplets may appear as uniformly round bright globules of various sizes under high power brightfield. Oil droplets from catheter lubricants may be confused with cells, especially red cells. Lipid material from vaginal creams also forms droplets in urine.

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Trichomonas versus White Cells

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.

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Starch Granules

Some starch granules from dusting powders have faint concentric striations, others do not. External contaminants (ie, those that enter the urine specimen during collection, transportation, or while being examined on the slide) must be differentiated from crystals and other clinically significant findings.

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Sperm

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

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Transfusion Reactions
Iron Overload

A unit of Red Blood Cells 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 arrhythmia. 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 that binds to iron and helps remove it through the urine or feces.

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Preliminary Laboratory Investigation

When the laboratory receives notification of a transfusion reaction, the first step is a clerical check. The clerical check should be performed as soon as possible to identify any possible ABO incompatibility. The technologist will compare the component bag, label, paperwork, and patient sample and look for errors. If an error is found, the physician must be notified. Once the post-transfusion sample is received, the sample should be examined for the presence of hemolysis. Both the pretransfusion sample and post-transfusion sample can be compared. Destruction of red cells and release of free hemoglobin will result in a pink to red supernatant. Pink or red colored serum may indicate intravascular hemolysis. The patient's serum may appear icteric if the hemolytic process is extravascular. The ABO testing must be repeated on the post-transfusion specimen as well. Examination of a post-reaction urine sample made aid in the diagnosis of acute hemolysis. Free hemoglobin in the urine indicates intravascular hemolysis. A direct antiglobulin test (DAT) must be performed on the post-transfusion sample. An EDTA lavender top tube is the required specimen type. If the DAT is positive on the post-transfusion sample, then one should be performed on the pretransfusion sample. If the pretransfusion DAT is negative and the post-transfusion is positive, the presence of incompatible red cells should be suspected. All findings must be reported to the supervisor or medical director, who may request additional tests.>

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Additional Testing

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 pretransfusion 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 pretransfusion 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 that affected the blood should be suspected, such as inappropriate warming or a faulty infusion pump. If bacterial contamination is suspected, the unit should be cultured. A positive culture indicates a reaction due to bacterial contamination.

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Procedure for a Suspected Adverse Reaction

Adverse reactions after transfusion of blood components must be evaluated promptly. Most serious reactions occur within the first 15 minutes of starting a transfusion. Continuous monitoring allows reactions to be discovered in a timely manner. The transfusionist must be able to recognize the symptoms of a transfusion reaction and know the appropriate steps to take when one occurs. The first critical step is to stop the transfusion immediately, but keep the patient's line open with saline. The physician should be contacted immediately for instructions regarding patient care. The transfusion service must be notified of the reaction. They will usually provide instructions on proper documentation of the reaction, and the return of any remaining component and/or tubing. The appropriate patient samples are to be sent to the laboratory and usually include blood and urine. The transfusionist must be sure to follow all hospital policies.

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Clinical Signs and Symptoms

Although there is no consistent clinical picture of an acute hemolytic transfusion 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.

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Management and Prevention

The first component of therapy is to stop the transfusion immediately. Vital signs must be closely monitored. Management involves treatment of hypotension and disseminated intravascular coagulation (DIC). It is essential to maintain blood volume and adequate renal blood flow. Diuretics, substances that increase urine output, may be administered. If the patient enters renal failure, dialysis must be initiated rapidly. It is impossible to prevent all hemolytic transfusion reactions. The purpose of pre-transfusion compatibility testing is to decrease the probability of a hemolytic transfusion reaction by performing ABO/Rh testing, detecting and identifying alloantibodies, and crossmatching compatible blood. Human error, the most common cause of hemolytic transfusion reactions, cannot be completely eliminated. Steps must be taken to reduce the possibility of human error in identification of patient samples, donor units, and recipients. Each person involved in the transfusion process, from collection of the blood sample to administration of the donor unit, must carefully adhere to each step outlined in the standard operating procedures. All appropriate protocols must be followed. Some examples are:Technologist checks blood sample to ensure proper labeling. Patient's previous transfusion records are examined and all transfusion testing is performed correctly and accurately. Technologist ensures correct unit is released from the blood bank. Transfusionist ensures the recipient is correctly identified.There must be a mechanism in place to train and assess all personnel involved in the transfusion process.

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White Cell and Platelet Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
Multiple Myeloma

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.

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