Urine Information and Courses from MediaLab, Inc.

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.

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 voiding, the specimen should be refrigerated immediately. Allow the specimen to return to room temperature before testing. The 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.

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. The affected tests should not be reported from the dipstick. It would be necessary to use an alternative method of testing if available.

Due to the wide range in urine pH values in healthy individuals, pH results must be evaluated in conjunction with the 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

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 increases the urine pH. If there is a delay before performance of the test, the sample should be refrigerated. This will: Prevent urease-producing organisms, such as Proteus and Pseudomonas, from converting urine urea to ammonia, which results in an increased pH. Prevent loss of CO2 which increases pH to the alkaline range.The "run-over" phenomenon may occur if excess urine remains on the strip. The protein area, adjacent to the pH area, contains an acid buffer which may "run-over" the pH portion resulting in an acid reading on a neutral or alkaline urine.

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.

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.

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.

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.

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.

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.

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

Testing for ketone bodies is based on a nitroprusside reaction. Acetoacetic acid reacts with sodium nitroferricyanide and glycerine 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.

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.

Ketone bodies are usually absent in urine. 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. 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. It is also an early indicator of insulin dosage problems in juvenile diabetes or in diabetics experiencing other medical problems. Electrolyte imbalance and dehydration occur when ketones accumulate in the blood. If these conditions are not corrected, the patient may develop acidosis and ultimately diabetic coma. 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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

Specific gravity 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 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 result when the pH is alkaline. Most dipstick readers, however, will automatically adjust the specific gravity reading for pH. A specific gravity reading higher than the reagent strip range would need to be measured by another method, and may require dilution.

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.

Normal urine contains very little protein, usually less than 10mg/dL, and the major serum 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 which 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.

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:

The pH of the urine must be approximately 7 or less in order to avoid false negatives with this test. If the pH exceeds 7, add 33% acetic acid dropwise to acidify the specimen to a pH of 7. If the specimen is cloudy, it should be centrifuged before SSA reagent is added. Daylight or fluorescent light is recommended when interpreting results. Quality control checks should be performed by testing known negative and positive specimens or controls.

Bilirubin is formed as a result of the breakdown of hemoglobin from erythrocytes in the reticuloendothelial system. It becomes bound to albumin and transported through the blood to the liver. This free or unconjugated bilirubin is insoluble in water and cannot be filtered through the glomerulus of the kidney. In the liver, bilirubin becomes conjugated with glucuronic acid to form bilirubin diglucuronide. This conjugated bilirubin, which is also called direct bilirubin, is water soluble and is excreted by the liver through the bile duct and into the duodenum.

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

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.

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 Pyridium. Because of this it is important to verify positive bilirubin results with a confirmatory test such as the Ictotest®.

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 quickly as possible when received in the laboratory. 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.

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, called hyperglycemia. Diabetes mellitus is the most common disease that causes hyperglycemia. However, stress, obesity, brain injury, myocardial infarction, hyperthyroidism, pregnancy, and a lowered renal threshold due to kidney damage can all cause glycosuria.

Prompt testing (within one hour of collection of the urine sample) 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.

Testing pediatric urine specimens for reducing substances other than glucose is a policy that should be implemented in the urinalysis laboratory. The maximum age for this testing is defined by each laboratory and is usually based on consultation with the pediatric clinical staff. The policy that is implemented in most laboratories is to test urine specimens for other reducing substances if the glucose test on the reagent strip is negative and the urine specimen is from a child below the age of one. Verify the policy for your own laboratory because the cutoff age for testing may be different.

Place 2 drops of urine in a test tube.

“Ketone bodies” is a generic term which refers to acetoacetic acid (diacetic acid), acetone, and beta-hydroxybutyric acid. Screening procedures used to detect ketonuria do not react with all ketone bodies. Since all three of the ketone bodies will be present in the urine and are equally significant, it is sufficient to detect an increase in any one or two of the ketone bodies. Most procedures, including Acetest®, measures acetoacetic acid and acetone but not beta-hydroxybutyric acid.

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

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)

This program is intended to provide guidance and training to those individuals who will be conducting Department of Transportation (DOT) 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 you see in this program conflicts with the federal regulations (49 CFR 40), the federal regulations prevail 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 pink copy of the CCF or refuses to initial the security strips.

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.

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 Employee Representatives (DER) to contact about any problems or issues that may arise during the collection process.

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.

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.

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 contact the Designated Employer Representative (DER) about the refusal as soon as is practical.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 contact the DER about the refusal as soon as is practical.

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.

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 August 25th, 2008.

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.

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.

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.

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.

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.

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.

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.

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, 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 of seen by phase-contrast microscopy.

Urine specimens 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 "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.

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.

Mix urine specimen well, and transfer 10-15 ml of urine to conical centrifuge tube. Centrifuge at 1500 rpm for 10 minutes. Decant supernatant, and resuspend sediment in 0.5-1.0ml of residual specimen. Place a drop of concentrated sediment on a glass slide and coverslip.

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 just above center of the image to the right 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.

To review the characteristics of the cellular elements that can be seen in urine, study the following table. Cell Significance Epithelial - Squamous Must be differentiated from other cells. Epithelial - Cuboidal or Renal Tubular Increased numbers indicate tubular necrosis, particularly important in renal graft rejection. Tubular damage caused by viral or bacterial infections. Epithelial - Transitional or Caudate Not 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. Yeast Can be confused with RBCs unless they are budding. Yeast can be seen in diabetes mellitus or in patients with vaginal moniliasis. Bacteria May be a contaminant unless WBCs are present.

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.

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

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). Papancolaou 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.