General Laboratory: Urinalysis: Testing Online Training

Urinalysis is a reliable laboratory procedure for ruling in or ruling out many medical conditions and diseases.

Recognize how examinations of urine are performed and how to identify formed elements in urine sediment Identify how tests typically included in a chemical analysis of urine provide clinicians with valuable information to help detect metabolic and kidney disorders Identify how to perform a physical analysis of urine color, clarity, odor, and volume Identify the principles of tests included on urinalysis test strips, as well as list common alternative methods, as applicable Welcome to the Clinical Urinalysis Testing course. After completing this course, you’ll be able to: Select Next to continue. If your system reports Liquid Level Sense errors from either your Dilution Probe (DPP) or your Reagent Probes (RPP1, RPP2) or you receive repeated Reagent Probe errors during a wash, here are some areas you should check. Select the  link below to learn more about troubleshooting Liquid Level Sense errors. The first component of a routine urinalysis is to perform a  physical analysis of the urine. Observing urine color, clarity, odor, and  volume can provide useful clues to the presence of many substances.   Tests Performed during a Physical Analysis Learn about the physical properties of urine in normal versus abnormal samples. TitleTextColorTab TitleTextNormal Urine The yellow or amber color of normal urine is caused by the yellow pigment urochrome.  Abnormal Urine Pigments produced by particular diseases may also cause the urine to change color. Some characteristic urine colors and causes include the following: A dark color may indicate concentrated urine A pale color may indicate dilute urine Bile pigments may produce a yellow-brown or greenish color Porphyrins produce a dark brown-red color upon standing Hemoglobin gives a reddish-brown color Melanins cause urine to turn a brown-black color upon standing Alcaptonuria is identified by urine that turns dark brown or black upon standing    Other Facts Some of the new automated urinalysis instruments are also capable of determining urine color.  ClarityTab TitleTextNormal Urine Normal, freshly voided urine is usually clear or transparent.  Abnormal Urine If the specimen is alkaline, it may have a cloudy or turbid appearance due to the presence of phosphates and carbonates. This cloudiness will usually disappear when the urine is acidified A pinkish turbidity frequently indicates the presence of urates Abnormal turbidity of urine may occur with urinary tract infections, but this is usually due to the alkalinity rather than the actual number of bacteria or leukocytes present Note: In about 10% of urine specimens, turbidity formed during refrigeration will not dissolve when the urine is brought to room temperature.  Other Facts Some of the new automated urinalysis instruments are also capable of determining urine clarity.  OdorTab TitleTextNormal Urine The smell of normal, freshly voided urine is believed to be due to the presence of volatile acids.  Abnormal Urine Urine that has been standing for a long time develops an ammonia-like odor, which is due to the decomposition of urea by bacteria The urine of people with diabetes mellitus may have a fruity odor due to the presence of ketones The urine of people with urinary tract infections may be foul-smelling, especially when the infecting organism is a coliform bacillus Innate metabolic disorders may also cause distinctive odors in urine including isovaleric acidemia, which produces a "sweaty feet" odor, and maple syrup urine disease, which was named for its odor Certain foods, such as asparagus, may produce a characteristic odor  Other Facts While urine may have many characteristic odors, as a rule these odors are not considered diagnostically significant, although there are exceptions.  VolumeTab TitleTextNormal Urine The normal volume of urine voided by an adult in a 24-hour period ranges from 750 to 2,000 mL, but averages about 1,500 mL. The amount voided over any period is directly related to: fluid intake temperature and climate amount of perspiration that occurs Children void somewhat smaller quantities than adults, but the total volume voided is greater in proportion to their body size.    Abnormal Urine Individuals can also produce too much or too little urine. Polyuria refers to an increase in the excretion of urine. It is a physiologic response to: increased fluid intake; the ingestion of diuretic medications; certain diuretic drinks, such as coffee, tea, and alcohol; chilling of the body; nervousness and anxiety; and intravenous infusion of fluids. Oliguria refers to decreased urinary output (i.e., less than 200 mL/24 hours). The extreme form, anuria refers to a total lack of urine. Oliguria occurs when there is excessive loss of body fluids as in vomiting and diarrhea; kidney shutdown through inflammation (nephritis), poisoning, or in cardiac insufficiency; mechanical obstruction of the urinary flow; decreased fluid intake; increased ingestion of salt; and excessive perspiration.  Other Facts Polyuria occurs in several disease states, particularly in diabetes mellitus and diabetes insipidus. It is a symptom of chronic kidney disease and has been noted in patients with certain tumors of the brain and spinal cord, as well as acromegaly and myxedema. Polyuria may indicate the loss of concentrating ability by the kidneys. When complete, select the X in the upper-right corner to close the window and continue   The second component of a routine urinalysis is to run a series of chemical tests. Most of the chemical tests performed during a urinalysis involve using chemically impregnated reagent strips or urinalysis test strips. Test strips allow laboratories to perform a single test and obtain results for a number of different test parameters. In this section, you will review how tests typically included in a chemical analysis of urine provide clinicians with valuable information to help detect metabolic and kidney disorders. Note: Chemical tablets, selectively treated slides, and simplified culture tests are also available for special determinations. Tests Performed during a Chemical Analysis I Learn about specific gravity, pH, osmolality, protein, and albumin tests. Tab TitleTextSpecific GravitySpecific gravity measurements reflect the relative degree of concentration or dilution of the specimen, which, under normal circumstances, correlate to the concentrating and diluting abilities of the kidney. Specific gravity measurements are necessary to interpret most routine urinalysis tests. Specific gravity indicates the density of the urine by measuring the total solids in urine and is a number derived from the ratio of the weight of a given volume of urine to the weight of the same volume of water, under standardized conditions. Specific Gravity = Weight of Urine / Weight of Water Water has a specific gravity of 1.000. Since urine is a solution of minerals, salts, and organic compounds in water, the specific gravity of normal urine is greater than 1.000.pHSecretion of an acid or alkaline urine by the kidneys is one of the most important mechanisms the body uses to maintain a constant body pH. Urine's acidity is due primarily to acid phosphates, with only a minor portion contributed by organic acids such as pyruvic, lactic, and citric acids. These acids are excreted in the urine as salts, primarily sodium, potassium, calcium, and ammonium salts. The kidneys maintain normal acid-base balance primarily through the reabsorption of sodium by the renal tubules and the tubular secretion of hydrogen and ammonium ions in exchange. To maintain acid-base balance, the acidity of urine increases as the amount of sodium retained by the body increases. The alkalinity of urine increases if there is an excess of base or alkali in the body. OsmolalityUrine osmolality is important for understanding the concentrating ability of the kidney. It is useful in: Determining the differential diagnosis of hyper- or hyponatraemia Differentiating pre-renal from renal kidney failure For identifying and diagnosing diabetes insipidus ProteinMost proteins are too big to filter through kidney glomeruli. However, serum proteins can leak into the urine when glomeruli are damaged, causing proteinuria. Urine protein testing is used to assess kidney function and to help detect and diagnose early kidney damage and disease. Proteinuria may also reflect urinary tract or physiological conditions rather than intrinsic kidney disorders. In disease states, smaller proteins, such as albumin and alpha-1 globulin, are excreted more readily than larger proteins. Albumin constitutes between 60% and 90% of protein excreted in most disease states. Certain diseases are characterized by the excretion of specific globulins rather than by a diffuse proteinuria. The urine of people with multiple myeloma contains increased amounts of a low molecular weight globulin (Bence Jones protein).  AlbuminIn disease states, smaller proteins, such as albumin, are excreted into the urine more readily than larger proteins. Albumin constitutes between 60% and 90% of protein excreted in most disease states. In patients with diabetes, microalbuminuria (low levels of albumin in the urine) is one of the first signs of deteriorating kidney function. As kidney function declines, the amount of albumin in the urine increases. When complete, select the X in the upper-right corner to close the window and continue. Tests Performed during a Chemical Analysis II Learn about creatinine, P:C ratio, A:C ratio, glucose, and ketone tests. Tab TitleTextCreatinineThe value of the creatinine test is that it can be used to calculate P:C and A:C ratios that correct for varying urine concentrations. Creatinine is derived from the non-enzymatic dehydration of creatine in skeletal muscle. The amount of creatine per unit of muscle mass is constant, and thus the breakdown of creatine to creatinine is constant in health. It is this consistency that has enabled creatinine to be used to correct for urine concentration and/or volume when the collection accuracy of a 24-hour urine specimen for protein excretion is in question.P:C RatioProtein-to-Creatinine Ratio (P:C Ratio) The P:C ratio test permits the estimation of the 24-hour protein excretion. Testing for proteinuria and obtaining a P:C ratio result is a useful tool for the early detection of kidney disease. Note: Accurately timed 24-hour urine specimens have also been used to express protein excretion in units of g/min. However, 24-hour urine specimens are difficult to accurately collect, necessitating a means to correct for urine concentration and/or volume when the collection accuracy is in doubt.A:C RatioAlbumin-to-Creatinine Ratio (A:C Ratio) The A:C ratio test permits the estimation of the 24-hour albumin excretion. Testing for the A:C ratio on random urine samples has been found to be as valid an indicator of microalbuminuria as a timed 24-hour sampling. The A:C ratio detects very low levels of albumin in the urine (microalbuminuria), and is therefore most appropriate for detecting early kidney damage in people with diabetes. Note: Accurately timed 24-hour urine specimens have also been used to express albumin excretion in units of g/min. However, 24-hour urine specimens are difficult to accurately collect, necessitating a means to correct for urine concentration and/or volume when the collection accuracy is in doubt.GlucoseThe presence of detectable amounts of glucose in urine is known as glycosuria. Diabetes mellitus is the chief cause of glycosuria, although the condition may be either benign or pathological. Other non-glucose reducing sugars, such as lactose, fructose, galactose, and pentose, may also be found in urine under certain conditions.  KetonesDiabetes mellitus is the most important disorder in which ketonuria occurs. Ketonuria also accompanies the restricted carbohydrate intake that occurs in association with fevers, anorexia, gastrointestinal disturbances, fasting, starvation, cyclic vomiting, pernicious vomiting of pregnancy, and cachexia. It also occurs following anesthesia and as a result of certain neurological disorders. When complete, select the X in the upper-right corner to close the window and continue.   Tests Performed during a Chemical Analysis III Learn about bilirubin, blood, nitrites, leukocyte esterase, and urobilinogen tests. Tab TitleTextBilirubinBilirubin in the urine is a sign of hepatocellular disease or intra- or extra-hepatic biliary obstruction. Bilirubin is formed by the breakdown of hemoglobin in the reticuloendothelial cells of the spleen and bone marrow. It is linked to albumin in the bloodstream and transported to the liver. Two forms of bilirubin are indirect bilirubin and direct bilirubin. Direct bilirubin can be excreted by the kidneys, although normally its level in the blood is not high enough to cause significant amounts to appear in the urine.  BloodAlthough protein in urine is the most important indication of kidney dysfunction, the presence of blood in urine is also a sign of damage to the kidney or urinary tract. Blood may appear as intact red blood cells (hematuria) or as free hemoglobin (hemoglobinuria).  NitriteThe nitrite test provides an indirect method for early detection of significant bacteriuria. The most common infecting organism of the urinary tract is Eschericia Coli, and less common are Klebsiella, Proteus, and Pseudomonas. These infecting organisms contain reductase enzymes that reduce nitrate in the urine to nitrite. Nitrite converters are generally gram-negative bacteria and gram-positive organisms may not be detected.Leukocyte EsteraseThe leuckocyte esterase test detects esterase released from the granules of neutrophilic leukocytes into the urine. The detection of leukocyte esterase indicates bacteriuria and is an indirect test for urinary tract infections. When WBCs are found in the urine sediment (pyuria), experienced technologists will look harder to find bacteria and correlate other indications of urinary tract infections, such as high pH or positive nitrite tests. It is usually agreed that the combination of positive results for both nitrite and leukocyte esterase is a good indicator of the need to perform a microscopic examination of the urine sediment for bacteria. Often, however, the combination of two positive chemical tests leads directly to confirmation of bacteriuria by microbiological culture testing.  UrobilinogenDetermination of urinary urobilinogen is a useful procedure in routine urinalysis since it serves as a guide in detecting and differentiating liver disease, hemolytic disease, and biliary obstruction. Sequential determinations also assist in evaluating progress of the disease and response to therapy. When complete, select the X in the upper-right corner to close the window and continue.   Chemically impregnated reagent strips or urinalysis test strips have virtually replaced older, more cumbersome chemical urinalysis test methods. Select the links below to learn about the chemical reactions that occur in the reagent areas on urinalysis test strips, as well as alternative test methods, as applicable. Note: The specific chemical reactions provided here are examples for  educational reference only. Please refer to your manufacturer's package  insert for specific information. Download and print information about alternative methods laboratories use to measure specific gravity. Test Method Principles Learn more about test method principles Checklist TitleChecklist TypeChecklist ContentSpecific GravityHTML Dip-and-read strips are the most common method of estimating urine specific gravity. The chemical reaction of the specific gravity reagent area on urinalysis test strips involves three primary ingredients, which are impregnated into the reagent paper: a polyelectrolyte (polymethylvinyl ether/maleic acid, partially neutralized), bromthymol blue indicator, and buffers. The principle of the colorimetric reaction is based on a pKa change of certain pre-treated polyelectrolytes in relation to the ionic concentration. In the specific gravity reagent area of the urinalysis test strips with multiple parameters, the polyelectrolyte is sensitive to the number of ions in the urine specimen. When the concentration of the electrolytes increases (high specific gravity) in the urine, the pKa of the polyelectrolyte in the urinalysis test strip is decreased. Thus, the pH decreases. The bromthymol blue indicator changes color from blue-green to green to yellow-green, indicating the pH change caused by increasing ionic strength (increasing specific gravity) and is empirically related to specific gravity values. Select each checkbox to learn more about test method principles. CreatinineHTML   The colorimetric urinalysis test strip is based on the peroxidase-like activity of copper-creatinine complexes. With  3,3',5,5'-tetramethylbenzidine (TMB) and diisopropyl benzene dihydroperoxide  (DBDH), the peroxidase-like activity of copper-creatinine complexes is measured. BiblirubinHTML Bilirubin Test Strip Method For the determination of bilirubin: The reagent area is impregnated with stabilized, diazotized 2, 4-dichloroaniline which reacts with bilirubin in urine to form a brownish-to-purplish-colored azobilirubin compound. The urinalysis test strip is dipped into fresh, uncentrifuged urine, tapped to remove excess urine, and, after a 30-second wait, compared to the color chart on the urinalysis test strip bottle. Ictotest Confirmation All positive reactions, or atypical reactions, need to be confirmed by using Ictotest® Reagent Tablets. Place 10 drops of urine on one special test mat. If bilirubin is present in the specimen, it will be adsorbed onto the mat surface. Place an Ictotest Reagent Tablet on the moistened area of the mat Flow two drops of water over the tablet. When elevated amounts of bilirubin are present in the urine specimen, a blue to purple color forms on the mat within 60 seconds. The rapidity of the formation of the color and the intensity of the color development are proportional to the amount of bilirubin in the urine. Normal amounts of bilirubin in the urine give a negative result. The smallest concentration of bilirubin reliably detected by this method is 0.05 to 0.1 mg/dL. An orange to red color may indicate the presence of Pyridium metabolites or azo dyes from other drugs. Note: These convenient, more specific tests have replaced the older oxidation procedures. pHHTML Test Strip Method The pH portion of urinalysis test strips is impregnated with two separate indicators, methyl red and bromthymol blue. The indicators provide a wide spectrum of color changes, from orange to green to blue, in the pH range of 5 to 8.5., and The color change is compared to a standardized color chart on the bottle label, which shows the pH values ranging from 5 through 8.5. Note: Since urine pH is almost always measured as a part of the more complete urinalysis, it is advantageous to use a urinalysis test strip with multiple parameters to simultaneously measure pH and check the urine for several other components. When more exact determinations are needed, a pH meter should be used.  Specimen Collection The accurate measurement of urinary pH can only be done on freshly voided specimens, as urine may become alkaline upon standing due to loss of carbon dioxide and the conversion of urea into ammonia by certain bacterial organisms. Urine samples that will not be tested within one hour should be refrigerated. P:C RatioHTML   A creatinine test pad on urinalysis test strips allows for semi-quantitative measurement of the P:C ratio as a means of improving strip result correlation to actual analyte excretion rates. The ratio allows for the use of single-void specimens in the discrimination of normal and abnormal levels of protein. The P:C ratio is calculated from the protein and creatinine results. Chemistry analyzers calculate the results automatically. Both methods provide clinicians with a convenient and efficient manner to obtain all results simultaneously. Blood (Hemoglobin)HTML Test Strip Method The urinalysis test strip method is the simplest and most direct test for the presence of blood in urine. The blood reagent area on urinalysis test strips is impregnated with tetramethylbenzidine and buffered organic peroxide. The composition forms a green to dark blue compound when hemoglobin catalyzes the oxidation reaction of tetramethylbenzidine with a peroxide. If read visually, the urinalysis test strips are compared with a color chart 60 seconds after the strip is dipped into the urine. The color ranges from orange through green, indicating negative, non-hemolyzed trace, non-hemolyzed moderate, hemolyzed trace, small (1+), moderate (2+), and large (3+) amounts of blood. Occult Blood Most urinalysis test strips with four or more reagent areas include a test for occult blood. The test is usually capable of detecting 0.015 to 0.062 mg/dL of free hemoglobin, which is equivalent to 5 to 20 intact red blood cells per microliter of urine. ProteinsHTML Test Strip Method The colorimetric reagent strip test is based on the ability of proteins to alter the color of some acid-base indicators without altering the pH, known as the "protein error of indicators" principle. When an indicator, such as tetrabromphenol blue, is buffered at pH 3, it is yellow in solution without protein but, in the presence of protein, the color will change to green and then to blue with increasing protein concentrations. In visual reading, protein is determined simply by dipping the strip into well-mixed uncentrifuged urine and comparing the resulting color with the chart provided on the reagent strip bottle. Other Test Methods A number of simple, semi-quantitative tests and more complex quantitative tests are available for the determination of all proteins in urine. Specific methods are used for the detection and quantification of albumin, globulins, Bence Jones protein, and others. The majority of these methods, with the notable exception of the simple colorimetric reagent strip test, depend on the precipitation of protein as the basis for quantitative determinations. While any significant amount of Bence Jones protein will be detected by colorimetric or turbidimetric screening tests, the simplest method for screening is gradual heating of a urine specimen to the boiling point. When this protein is present, a precipitate will first appear, then dissolve as the urine is further heated. The presence of large amounts of other proteins or phosphates decreases the accuracy of this test. These interfering proteins can usually be removed by cooling the heated urine to room temperature, filtering, and repeating the heating process on the filtrate. A:C RatioHTML   A creatinine test pad on urinalysis test strips is used to calculate A:C ratio as a means of improving strip result correlation to actual analyte excretion rates. The ratio allows for the use of single-void specimens in the discrimination of normal and abnormal levels of protein. The A:C ratio is calculated from the albumin and creatinine results. Urine chemistry analyzers calculate the results automatically. NitriteHTML   The nitrite area of urinalysis test strips has a chemical compound, para-arsanilic acid, impregnated into the strip. This reagent forms a diazonium compound with nitrite which, in turn, couples with a tetrahydro quinoline derivative to produce a pink-colored compound. AlbuminHTML The flow chart shown here by the American Diabetes Association can be used as a guide to microalbuminuria testing. GlucoseHTML Test Strip Method Enzymatic tests based on the action of glucose oxidase on glucose is used for urinalysis test strips. Enzymatic glucose oxidase tests, as applied to urine, are specific for glucose. In these tests: Glucose oxidase catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide. The peroxide, in the presence of peroxidase, oxidizes an indicator that produces a color change. Other sugars, such as lactose, fructose, galactose, and pentose, are not substrates for glucose oxidase and, therefore, do not react with this test. The resulting glucose color reaction is compared to a six-block color chart ranging from blue, indicating less than 0.1% concentration of glucose, to brown, indicating 2.0% or more.  Other Test Methods Reduction tests based on the reduction of certain metal ions by glucose is another method used to test urine glucose. The reduction of metallic ions, such as Cu++, is non-specific for glucose because the reaction may be initiated by any reducing substance present in the urine, such as large quantities of creatinine, uric acid, ascorbic acid, or some other reducing sugar. While non-carbohydrate components seldom interfere with results, some interference occasionally occurs in concentrated urine. The non-specificity of this test has advantages and disadvantages. The advantage is that it detects important sugars, such as galactose and lactose; the disadvantage is that it detects reducing substances other than glucose. Detecting Non-Glucose-Reducing Sugars Galactose can be detected with reagent tablets and identified by paper chromatography and by the galactose oxidase test. Some pediatricians screen infants for galactosemia using reagent tablets and urinalysis test strips to detect the presence of non-glucose reducing substances (positive tablet, negative urinalysis test strip). Lactose in urine can be detected by reagent tablets. Identifying the sugar as lactose, although not a routine procedure, can be done by paper chromatography. Fructose can be detected with reagent tablets. It can be identified by Selivanoff's test and by paper chromatography, neither of which is a routine procedure. Pentose can be detected with reagent tablets. Identification of a pentose can be accomplished by chromatography. Leukocyte EsteraseHTML The leukocyte esterase test on urinalysis test strips detects the esterase released from white blood cells, for example polymorphonuclear leukocytes (mostly neutrophils). The principle of the reaction is that the enzyme splits an ester to form a pyrrole compound that reacts with a diazo reagent to form a highly colored azo dye. The intensity of color is proportional to the amount of enzyme in the urine and, in turn, proportional to the number of white blood cells in the urine. KetonesHTML Test Strip Method The ketone reagent area on the urinalysis test strip is impregnated with sodium nitroprusside and alkaline buffers. The strip is dipped into fresh urine, and then the underside of the strip is dragged along the inside of the container to remove excess urine. The reagent pad color is compared to the color chart after exactly 15 seconds. The chart has six color blocks ranging in color from buff to lavender and maroon and indicating negative, trace (5 mg/dL), small (15 mg/dL), moderate (40 mg/dL), or large (80 mg/dL) and (160 mg/dL) concentrations of ketone. The test is sensitive only to acetoacetic acid. It does not react with beta-hydroxybutyric acid or acetone. Compounds that contain sulfhydryl groups, such as mesna (2-mercaptoethane sulfonic acid) may cause false positive results or an atypical reaction. Other Test Methods While specific tests do exist for the determination of each of acetoacetic acid, acetone and beta-hydroxybutyric acid, they are not widely used because they tend to be more cumbersome, less reliable, and less sensitive. UrobilinogenHTML Test Strip Method The urobilinogen reagent area of urinalysis test strips is impregnated with para-diethylaminobenzaldehyde and an acid buffer solution. It reacts with urinary urobilinogen, porphobilinogen, and para-aminosalicylic acid to form colored compounds. The strip is dipped into fresh, uncentrifuged urine, collected without preservatives. It is then removed and, after exactly 60 seconds, the color reaction is compared to the color chart. The five color blocks provided on the urinalysis color chart range from peach to pink, representing 0.2, 1, 2, 4, and 8 mg/dL. The first two color blocks, 0.2 and 1 mg/dL, are within the normal range of values for urobilinogen. The remaining three color blocks indicate elevated values. This test will detect the absence of urobilinogen.  Sample Collection A freshly voided urine specimen is necessary for the test, preferably a sample collected over a two-hour period in the early afternoon when urinary urobilinogen excretion is thought to be at the highest rate for the day. Inhibitors Formaldehyde, which may be used as a preservative, can inhibit this reaction, causing falsely lowered results. No other substances are known to clearly inhibit the reaction. Drugs containing azo dyes will have a masking effect on the urobilinogen area. When complete, select the X in the upper-right corner to close the window and continue. The Unified Fluidics Circuit, or UFC, is the "heart" of the ADVIA 2120i. Here, the sample is aliquotted and then all of the reactions occur. After the reactions are complete, the sample is shuttled to the syringe for delivery to the flowcell. You will learn the various components of the UFC. Select the link below to learn more about UFC components. Standardized results can be achieved by processing the reacted urinalysis test strips with special instrumentation, although you can also read test strips visually. The procedure listed below represents the general procedure used. However, to obtain reliable results the procedure listed on the package insert or in the manufacturer's manual must be followed exactly. Collect a fresh urine specimen in a clean, dry container. Mix well immediately before testing. Remove one strip from the bottle and replace the cap. Completely immerse the reagent areas of the strip into the urine and remove immediately to avoid dissolving out the reagents. Note: If there is insufficient urine available to completely immerse all of the reagent pads, tip the container so that the urine reaches a sufficiently high level to be able to wet all reagent areas. Start timing. While removing the strip from the urine container, run the edge of the strip against the rim of the urine container to remove excess urine. Hold the strip in a horizontal position to prevent possible mixing of chemicals from adjacent reagent areas and/or contaminating the hands with urine. Compare the reagent areas to the corresponding color chart on the bottle label at the times specified. Hold the strip close to the color blocks and match carefully. Avoid laying the strip directly on the color chart, as this will result in the urine soiling the color chart. Select Next to continue.   Perform a microscopic examination of urine sediment to detect elements, such as cells, casts, crystals, bacteria, parasites, and artifacts. This information can provide: evidence of kidney disease as opposed to lower urinary tract infection indication of the type and activity of a kidney lesion or disease condition. Microscopic examination can also serve as a confirmatory test. The microscopic results and urine chemistry results should be checked against each other. Discrepancies should be explained before reports are issued.   Learn More: Screening Urine Samples Learn why some laboratories screen urine samples. Only cloudy urine specimens or specimens that show positive strip results for blood (RBCs), white blood cells (WBCs), nitrite, or protein are the most likely to show anything significant with the microscopic examination. Some laboratories have reduced the number of microscopic urine sediment examinations performed by first evaluating the urine appearance and urinalysis test strip results. These clinicians have determined that urine specimens may not require further testing unless an abnormal result is obtained for one or more of several urinalysis test strip parameters, such as blood, protein, leukocytes, nitrite, or clarity. This concept is often referred to as a "screen" or "sieve." This procedure is intended to eliminate the performance of labor-intensive microscopic examinations on specimens that tend to have a low yield of abnormal microscopic findings. Of course, clinicians must ultimately determine whether this testing protocol is appropriate for their particular laboratory situation and patient population. When complete, select the X in the upper-right corner to close the window and continue. It is vital that a standardized procedure be used to prepare the sample for examination to ensure consistency between operators, and from one institution to another. While the first-morning specimen is usually the preferred specimen, other specimens can be used The urine specimen should be examined as soon as possible (less than two hours after collection), as the formed elements (cells, casts, etc.) begin to lyse upon standing If the specimen cannot be examined within two hours, it must be refrigerated or otherwise preserved Refrigeration may cause the precipitation of amorphous urates or phosphates (acid or alkaline urines, respectively) that can make it hard to see formed elements in the microscopic examination. Warming of the urine specimen to room temperature will re-dissolve the amorphous urates. The amorphous phosphates require the addition of a weak acid (acetic acid) for them to go back into solution Select Next to continue.   One way to standardize the microscopic examination is by using a complete test system that has special test slides that come complete with cover slips and a grid pattern on the slide so that the number of cells/µL of urine can be reported. The following is an example of a standardized procedure. Start with the same volume of fresh urine to be concentrated (12 mL) Centrifuge for a constant time (5 minutes), at a constant relative centrifugal force (RCF) of 400 G Decant or aspirate the supernatant to leave a standard volume of concentrate (0.5 mL) Examine a standardized volume of urine in a standardized slide chamber with a set depth, first under low power and then high power magnification Report the number of elements/µL of urine, based on a factor established for the specific test system. Note: When it is not a standardized determination, the actual number of cells counted should be interpreted with caution. Select Next to continue. Most microscopic examinations of urine sediment are made  under brightfield illumination. There are also instrument  systems that automate sediment analysis. Microscopic Examination Techniques Learn several techniques that can be used to examine urine sediment. Tab TitleTextBrightfield This standard examination should start with examination under "low power" magnification (10x objective and 10x eyepiece [100-x]), with contrast achieved by varying the opening of the iris diaphragm. This permits the observation of larger elements such as casts, especially hyaline casts, mucus, and some cells. The specimen is then examined with a 40x objective and 10x eye piece (400x or high power). Phase ContrastThis type of microscopy is particularly useful in identifying hyaline casts, mucus, cells, and bacteria that may be difficult to see using brightfield illumination.Interference ContrastInterference Contrast Illumination This method is not widely used, but it offers the benefits of phase contrast, plus showing the elements in 3D.Polarized LightPlain or compensated polarized light is ideal for assisting in the identification of crystals, starch, fat, and fibers.Automated Systems Instrument systems that automate sediment analysis of formed elements work on principles of flow imaging or flow cytometry to identify and quantify cells and other formed elements. Certain specimens with casts, crystals, or a large number of formed elements are appropriately flagged for visual review and/or the microscope. When complete, select the X in the upper-right corner to close the window and continue. Congratulations. You have completed the Sysmex CS-5100 System Overview online training course. Listed below are the key points that have been presented.  Take time to review the material before you proceed to the final quiz.  A variety of stains can be used to assist in the identification of various elements. These stains can include. Supravital stains, such as Sternheimer-Malbin stain or toluidine blue Lipid stains, such as the Sudan group Gram stain (to identify bacteria) Supravital Stains Learn about supravital stains. Elements in urine sediment take on the following characteristics when stained using a supravital stain: Leukocytes may appear unstained initially, or the nucleus may appear red-blue with red cytoplasm after a prolonged period of time Hyaline casts stain bright blue, while finely granular casts stain bright red Coarsely granular casts and "waxy" casts stain reddish violet RBC casts are outlined in shades of pink, while hemoglobin casts appear rust-brown Miscellaneous substances, such as oval fat bodies and yeast, will not stain Trichomonas will not stain or may appear bluish When complete, select the X in the upper-right corner to close the window and continue. What do you observe in normal urine sediment? Normal sediment is not free of cells or casts, but contains a limited number of formed elements, for example: The presence of one or two blood cells, one or two leukocytes, and a few epithelial cells is not necessarily abnormal An occasional hyaline cast may also be a normal finding The urine of mature females may normally contain large numbers of squamous epithelial cells from the vaginal walls Abnormal urine sediment, however, may contain many cells or casts that indicate a disease process. In this section, you will learn the significance of formed elements that may appear in urine sediment and how to identify them microscopically. Download and print a table that lists some crystals found in urine sediment and the physical characteristics associated with them. Crystals Learn about crystals observed in urine sediment. Tab TitleTextAll Crystals A variety of crystals may appear in the urine. The type and quantity of crystalline precipitate vary with the pH of the urine. Amorphous material is of little importance. While most crystals are non-pathological, some do indicate pathology. Crystals form in normal urine as the specimen cools. Examples of abnormal crystals include cystine, uric acid, and leucine and tyrosine. Crystals can be identified by their specific appearances and solubility characteristics. Cystine Cystine crystals indicate cystinuria, a condition in which cystine stones form in the kidney and cystinosis, an innate metabolic disorder in which cystine crystals are found in the urine, reticuloendothelial system, spleen, and eyes.   Uric Acid Uric acid crystals may appear in the urine in a variety of shapes and colors. They may appear as a result of pathology or metabolism. Uric acid may appear as needles, hexagonal shapes, rosette shapes, "whetstone form," or as rhombic plates. The crystals may appear colorless, yellow, or brown. Increased uric acid denotes increased purine metabolism. Uric acid crystals may be found in cases of fever, leukemia, some kidney tubular diseases, and gout. Leucine/Tyrosine Leucine and tyrosine are abnormal crystals occasionally seen in urine of people with liver problems. When there are severe liver problems, these amino acids are not metabolized. Tyrosine crystals appear as colorless fine needles and are usually grouped in clusters. When complete, select the X in the upper-right corner to close the window and continue. Organisms & Contaminants in Urine Sediment Learn about organisms and contaminants observed in urine sediment. Tab TitleTextBacteria Bacteria may be seen in the sediment as a result of either urinary tract infection or contamination of the specimen. The two causes cannot usually be distinguished by examination of the specimen, although the presence of large numbers of leukocytes, a positive nitrite test, and/or a positive leukocyte esterase test is suggestive of urinary tract infection. Bacilli are more easily recognized than cocci, which may be mistaken for amorphous crystals. A culture on a clean-catch specimen should be performed when in doubt.   Parasites The majority of parasites observed in urine are contaminants from fecal or vaginal material. A urinary tract parasite infestation may be associated with the presence of red blood cells, e.g., Schistosoma haematobium. Trichomonas vaginalis is the most frequently seen parasite in urine. It is a unicellular organism with anterior flagellae and an undulating membrane. The parasites may resemble flattened, ovoid epithelial cells, but are usually recognized by their swimming motions through the sediment, the movements of their flagellae, and the characteristic undulating membrane.   Yeast Yeast cells may be seen in the urinary sediment. Yeast cells (Candida albicans) may be indicative of urinary candidiasis, especially in people with diabetes mellitus. Frequently, yeast appears as a contaminant in the urine of females with vaginal candidiasis. Yeast cells are sometimes confused with red blood cells. They differ by being ovoid (rather than round), colorless, and variable in size. They may also frequently show budding. If in doubt, the addition of acetic acid to the sediment on the slide will lyse red blood cells but leave yeast cells intact. Large numbers of yeast with hyphae are suggestive of vaginitis.   Spermatozoa Spermatozoa are frequently seen in the urine following nocturnal emissions or sexual intercourse. Spermatozoa have oval bodies with long delicate tails. They may be mobile or stationary. Contaminants Cotton threads (1) and starch granules (2) shown here as well as hair, wood and wool fibers, and other contaminants must be recognized to ascertain that these substances do not represent any significant finding in the urinary sediment. When complete, select the X in the upper-right corner to close the window and continue. Casts Learn about about casts observed in urine sediment. TitleTextAll CastsTab TitleTextDescription Casts Overview Cast formation usually occurs in the distal convoluted tubule of the nephron. Casts may also occur in the ascending loop of Henle or the collecting duct Requirements for cast formation are an acid condition, high salt concentration, reduced urine flow, and protein Casts are named according to the inclusions contained in them, such as RBC cast, WBC cast, etc.  Microscopic Identification The appearance, size, and inclusion of a cast will offer incontrovertible evidence of the condition of at least one nephron of one kidney just prior to passage of the urine. Practically all casts have a hyaline matrix that may or may not contain inclusions, such as desquamated cells from the lining of tubules, white blood cells, or red blood cells.    RBCTab TitleTextDescription Red Blood Cell Casts Red blood cell casts indicate the presence of an acute inflammatory or vascular disorder in the glomerulus, causing kidney hematuria. They should always be regarded as pathological and may be the only manifestation of acute glomerulonephritis, kidney infarction, collagen disease, or kidney involvement in subacute bacterial endocarditis.  Microscopic Identification Red Blood Cell Casts Red blood cell casts form in three stages:   1. Presence of free red blood cells 2. Degenerating cells within a protein matrix 3. Homogeneous blood casts Any disease that alters the integrity of the glomerulus will alter the composition of the urine. Disease of injury to the glomerulus usually results in a leakage of RBCs and protein.  WBCTab TitleTextDescription White Blood Cell Casts White blood cell casts may be found in the urine of people with acute glomerulonephritis, nephrotic syndrome, or pyelonephritis. Since pyelonephritis may remain completely asymptomatic even though it is progressively destroying kidney tissue, careful examination of the urinary sediment for leukocyte casts is important. In some cases, it may be the only significant laboratory finding in an asymptomatic situation.  Microscopic Identification White Blood Cell Casts White blood cell casts are usually composed of many leukocytes in a cylindrical encasement and indicate kidney origin.  Epithelial CellTab TitleTextDescription Epithelial Cell Casts Epithelial cell casts are formed by fused desquamated tubular cells. Since the tubule is a living membrane, it is always replacing itself. Thus, the finding of an occasional kidney epithelial cell or clump is not unusual. However, in any disease producing damage to the tubular epithelium, the appearance of many epithelial casts may indicate excessive desquamation such as may occur in nephrosis, eclampsia, amyloidosis, and in the presence of poisoning with heavy metals and a variety of other toxins.  Microscopic Identification Epithelial Cell Casts Epithelial cell casts are formed by fused desquamated tubular cells. The degeneration of the discrete cellular casts into coarsely and finely granular material is purely a function of age and permits the inference that there has been stasis in the nephron.  HyalineTab TitleTextDescription Hyaline Casts Hyaline casts, formed of the gel of Tamm-Horsfall protein, may imply damage to the glomerular capillary membrane, permitting leakage of proteins through the glomerular filter. Such damage may be permanent or transient as a result of fever or the effects of posture (orthostatic, lordotic), emotional stress, or strenuous exercise. If a person exercises strenuously, the blood flow to the kidneys is reduced, as it is re-directed to the muscles. During this period, hyaline casts may be formed. When the exercise is over and the blood flow returns to normal, there is a "flushing out" of these casts into the urine. This "shower of casts" is usually non-pathological.  Microscopic Identification Hyaline Casts Hyaline casts are pale, colorless, occasionally refractile "cylinders." They are best seen when the intensity of the light is sharply reduced. These casts are formed from the gel of proteins that have presumably traversed the glomerular capillary membrane.  GranularTab TitleTextDescription Granular Casts When describing granular casts, the terms "coarsely granular" and "finely granular" are used to indicate the degree of degeneration that has occurred in the cellular inclusion, i.e., whether the cells have been broken down into coarse or fine particles. While an occasional granular cast may be found in normal individuals, numbers beyond occasional may indicate pyelonephritis. Granular casts are also found in chronic lead intoxication.  Microscopic Identification Coarse granular casts contain homogeneous, coarsely granular material. They are clear, colorless, and appear very dense. Coarse granular casts may represent the initial stages of degeneration of epithelial cell casts. These casts further degenerate into fine granular casts and terminate as waxy casts or fatty casts. Fine granular casts are differentiated from coarse granular casts by the presence of fine granular material.  WaxyTab TitleTextDescription Waxy Casts Waxy and fatty casts are associated with tubular inflammation and degeneration. The broad, waxy cast is formed in the collecting tubules when the urine flow through them is reduced. Both waxy and fatty casts are found in chronic kidney disease.  Microscopic Identification Waxy Casts Waxy casts are composed of a homogeneous, yellowish material. They are relatively broad, have a highly refractile outline, and appear very brittle. They are irregularly shaped, show characteristic clefts, and occasionally may have a "corkscrew" appearance. Broad casts (kidney failure casts) are two to six times as wide as ordinary casts. They are usually waxy, granular, or cellular. They are thought to appear in the collecting tubules as a result of markedly decreased urinary output, presumably due to severe kidney disease. When complete, select the X in the upper-right corner to close the window and continue.    Cells in Urine Sediment Learn about cells observed in urine sediment. TitleTextRBCsTab TitleTextDescription Red Blood Cells in Urine Sediment More than two to three red blood cells per high power field is an abnormal condition, as the presence of RBCs can indicate a variety of kidney and systemic diseases, including trauma to the kidney. Hematuria occurs with pyelonephritis, tuberculosis of the genitourinary tract, cystatitis, prostatitis, kidney calculi, kidney tumors, and other malignancies of the urinary tract, and hemorrhagic diseases, such as hemophilia RBCs may also appear following traumatic catheterization, passage of stones, contamination from menstrual blood, and strenuous exercise It is important to note that RBCs tend to lyse or dissolve in alkaline or dilute urine.  Microscopic Identification Red blood cells usually look like pale, light-refractive, bi-concave discs when viewed under high power magnification. They have no nuclei. Red blood cells seen in fresh, unstained sediment are pale in color. In urine that is not fresh, they are colorless "shadow cells." In concentrated urine, the red blood cells may be small and crenated. And in dilute urine, they are often large and swollen, and sometimes rupture to produce "ghost" cells. Red blood cells must be differentiated from yeast cells, urate crystals, and oil droplets. Yeast cells are usually ovoid and frequently show budding Ammonium biurate crystals occur in large quantities and in a great range of sizes Mineral oil droplets also vary greatly in their size and are more refractile and spherical  WBCsTab TitleTextDescription White Blood Cells in Urine Sediment The presence of large numbers of white cells or leukocytes (pyuria) usually indicates bacterial infection in the urinary tract. Pyuria may also be seen in acute glomerulonephritis. The cells are segmented neutrophils or polys. Large numbers of mononuclear cells (lymphs) in a patient with a kidney transplant may indicate early tissue rejection.  Microscopic Identification The predominant type of leukocyte appearing in the urine is the polymorphonuclear leukocyte. These leukocytes have segmented nuclei, are usually granular, and are approximately 1x as large as red blood cells. Certain neutrophils are larger than the usual leukocytes, and their cytoplasmic granules show Brownian movement. These cells are called granular motility or "glitter" cells. Originally they were thought to be pathognomic of pyelonephritis but are now thought to be a result of hypotonic urine. With white blood cells in the sediment, the urine should give a positive chemical test for leukocyte esterase.  Epithelial CellsTab TitleTextDescription Epithelial Cells in Urine Sediment Squamous epithelial cells frequently appear in normal urine. Large numbers of kidney epithelial cells, which are common in the urine of people with acute necrosis and necrotizing papillitis, may indicate active tubular degeneration. The image displayed here shows the origin of various epithelial cells that may appear in the urine. However, due to the osmotic, pH, and traumatic changes the cells undergo during passage through the genitourinary system, they rarely retain their original shape.  Microscopic Identification Kidney tubular epithelial cells (1) are round and slightly larger than leukocytes. Each contains a single large nucleus. Bladder epithelial cells (2) are larger than kidney tubular epithelial cells. They range in shape from flat to cuboidal or columnar. Squamous epithelial cells (3) are large flat cells with single small nuclei and a large cytoplasm. The majority of these cells are contaminants from the vagina or vulva, but some originate in the urethra. When complete, select the X in the upper-right corner to close the window and continue.    A broad range of urine chemistry analyzers automate the reading of urinalysis test strips, which helps standardize testing by eliminating operator-to-operator variability, as well as improves laboratory efficiency because read rates are as fast as seven seconds per test strip. Urine chemistry analyzers can be used in: Point-of-care settings, such as doctor offices, clinics, and hospital wards Laboratory settings, such as hospitals or private laboratories In most cases, these systems also interface with the Laboratory Information System (LIS) for data management and reporting. Select Next to continue.