Urinalysis : Chemical Examination and Interpretation.
A urinalysis is a group of physical, chemical, and microscopic tests performed on urine. The tests detect and/or measure several substances in the urine, such as byproducts of normal and abnormal metabolism, cells, cellular fragments, and bacteria.
Urine is produced by the kidneys. The role of the kidneys is to filter wastes out of the blood, help regulate the amount of water in the body, and conserve proteins, electrolytes, and other compounds that the body can reuse. Anything that is not needed is eliminated in the urine, traveling from the kidneys through ureters to the bladder and then through the urethra and out of the body.
Urine is generally yellow and relatively clear, but each time a person urinates, the color, quantity, concentration, and content of the urine will be slightly different because of varying constituents.
Many disorders may be detected in their early stages by identifying substances that are not normally present in the urine and/or by measuring abnormal levels of certain substances. Some examples include glucose, protein, bilirubin, red blood cells, white blood cells, crystals, and bacteria. They may be present because:
- There is an elevated level of the substance in the blood and the body responds by trying to eliminate the excess in the urine.
- Kidney disease is present.
- There is a urinary tract infection present, as in the case of bacteria and WBCs.
A complete urinalysis consists of three distinct testing phases:
- Visual/physical examination, which evaluates the urine's color and clarity, odor, and volume
- Chemical examination, which tests chemically for about 9 substances.
- Microscopic examination, which identifies and counts the type of cells, casts, crystals, and other components such as bacteria and mucus
Chemical Examination In Urinalysis
The chemical examination of urine can be performed using reagent strips, single-use narrow plastic strips with several reagent pads attached. Reagent strip analysis is also called biochemical, analyte, or “dipstick” analysis of urine.
A single reagent strip can test for as many as 10 different substances; other types of tests for only one or two substances or analytes.
Strips used in routine urinalysis usually include tests for urobilinogen, glucose, leukocyte esterase, and specific gravity.
Chemical testing by reagent strip is part of a routine urinalysis but can also be ordered separately, without the urine physical and microscopic examinations.
Chemical analysis of urine by reagent strip method is a CLIA-waived procedure.
The urine chemical test results can be an aid to diagnosis. The results of chemical analysis of urine provide information on the patient’s carbohydrate metabolism, kidney and liver function, and pH balance.
Each reagent pad on a reagent strip contains the chemicals required for a specific chemical reaction. Reagent strips are available in a variety of types.
Two common brands of urine reagent strips used in urinalysis are:
- The Multistix line (Siemens Medical, formerly Bayer Corp.) and
- Chemstrip line (Roche Diagnostics Corp.)
Reagent strips are used by dipping the strip into the urine and observing chemical reactions on each reagent pad within a specified time. The chemicals within the reagent pads react rapidly with substances in the urine causing color changes in the pads.
Color and timing guides for interpreting results are specific for each brand or type of strip and are provided by each reagent strip manufacturer.
In routine urinalysis, the reagent strips commonly used test 10 parameters:
- Leukocyte esterase
- Specific gravity
In this article, we shall cover the basic principles of the biochemical reactions involved in each of these 10 tests in urinalysis. However, because reagent strip manufacturers can use different biochemical detection methods in their strips, the manufacturer’s instructions must be consulted for specific chemical principles, correct procedure, and possible interferences for the test strip being used
The presence of detectable glucose in the urine is called glycosuria and indicates that the blood glucose level has exceeded the renal threshold for glucose (160 to 180 mg/dL). This condition can occur in diabetes mellitus and gestational diabetes.
The presence of glucose in the urine can be an indication that diabetes is not being well controlled.
The reagent strip is specific for glucose and will not react with other sugars. The glucose reagent pad contains the enzymes glucose oxidase and peroxidase plus a chromogen, a substance that produces color in the chemical reaction.
The enzymes react with glucose in the urine to change the color of the reagent pad.
The intensity of the color formed is proportional to the glucose concentration. Normal urine is negative for glucose by the reagent strip method.
Bilirubin is the primary bile pigment. Bilirubin is a hemoglobin breakdown product formed in the liver from senescent (old) red blood cells. When bilirubin is present in urine, it can indicate liver disease, bile duct obstruction, or hepatitis.
The test for bilirubin is based on coupling bilirubin with a diazonium salt in the reagent pad to form a purple-brown color. Direct light causes decomposition of bilirubin, so specimens should be protected from light until testing is completed to prevent false-negative results.
A positive reagent strip bilirubin test should be followed by the Ictotest, which is more sensitive and has fewer interferences. Normal urine contains no detectable bilirubin by the reagent strip method.
Bilirubinemia is the appearance of bilirubin in urine. Yellow foam forms when the sample is shaken
Ketones are immediate products of fat metabolism. When the body burns fat rather than sugar for energy, a group of molecules collectively called ketones are produced.
Ketones are a mixture of acetone, acetoacetic acid (also called diacetic acid), and beta-hydroxybutyric acid. Urine testing only detects acetoacetic acid not the other ketones, acetone or beta-hydroxybutyric acid.
In uncontrolled diabetes, starvation, or prolonged dieting or fasting, ketones are excreted into the urine, a condition called ketonuria. The ketones reagent strip pad is more sensitive for acetoacetic acid than acetone; beta-hydroxybutyric acid is not detected.
The ketone test is based on the reaction of ketones with sodium nitroprusside in the ketone reagent pad, causing a dark pink to maroon color to form. Because ketones evaporate at room temperature, urine should be kept tightly capped and refrigerated if it cannot be tested promptly. Normal urine is negative for ketones when tested by a reagent strip.
Ketonuria + glucose in urine may indicate diabetes mellitus.
The presence of blood in urine almost always indicated pathology because red blood cells are too large to pass through the glomerulus.
The presence of blood in the urine is called hematuria. Hemoglobinuria is a more specific term referring to the presence in the urine of hemoglobin from lysed red blood cells.
Hematuria can occur in conditions such as infection, bleeding in the kidneys, glomerular damage, kidney stone, tumor in the urinary tract, or trauma to the urinary tract from surgery or catheter insertion.
Blood cells can also contaminate urine as a result of menstruation
The blood reagent pad detects both intact red blood cells and free hemoglobin from lysed red cells. Myoglobin will also cause a positive reaction. In the presence of blood, color forms because of the peroxidase-like action of hemoglobin in the red blood cells reacting with chromogen and peroxide in the reagent pad.
The resulting color ranges from orange through green to dark blue and the degree of color change is proportional to the amount of blood present. Intact red blood cells can cause a spotty appearance on the reagent pad.
Always be cautious in women because of its a common finding in women because of menstruation
Normal urine is negative for blood by reagent strip method.
The pH is a measure of the degree of acidity or alkalinity of the urine. A pH below 7.0 indicates acid urine; a pH above 7.0 indicates alkaline urine. The pH of urine is affected by diet, medications, kidney disease, and metabolic diseases such as diabetes mellitus.
Urine samples from individuals on vegetarian diets usually have alkaline pH. Indicator dyes such as methyl red and bromothymol blue in the pH reagent pad form colors from yellow-orange for acid urine to green-blue for alkaline urine.
Normal, recently voided urine has a pH range of 5.0 to 8.0; first morning specimens should have an acid pH (5.0 to 6.0).
Phosphates will precipitate in alkaline urine and uric acid will precipitate in acidic urine.
Proteins are usually too large to pass through the glomerulus. Proteinuria usually represents an abnormality in the glomerular filtration barrier.
The condition in which an increased amount of protein is present in the urine is called proteinuria. Proteinuria is an important indicator of renal disease.
Proteinuria can be caused by urinary tract infection, commonly called UTI, and can also occur following vigorous exercise. Positive tests for protein in urine are due to the presence of albumin, the most abundant plasma protein.
Because the protein reagent pad is not a good detector of globulins, the protein sulfosalicylic acid turbidity test should be used to detect proteins other than albumin.
The reagent strip test for protein is based on the principle that proteins can alter the color of some acid-base indicator dyes without changing the pH. This reaction has been called “the protein error of indicators.”
The protein reagent pad is kept at pH 3.0 by incorporating a buffering dye such as tetrabromophenol blue. At the constant acid pH, the development of any green color on the reagent pad is due to the presence of protein (albumin) and is usually reported using a plus system (neg, trace, 11, 21,31, 41).
Colors range from yellow for negative to yellow-green or green for positive, depending on the amount of protein present.
Rarely, highly alkaline urine can produce a false positive protein test, because the alkaline urine pH can neutralize the acid pH in the reagent pad.
Normal urine is negative or contains just a trace of protein in pregnancy or after taking a lot of proteins by the reagent strip method.
urobilinogen is a bilirubin degradation product that is formed by the action of intestinal bacteria. Urobilinogen can be increased in hepatic disease or hemolytic disease.
The urobilinogen reagent strip test, based on the Ehrlich aldehyde reaction, contains chemicals that react with urobilinogen to form a pink-red color.
Urobilinogen is unstable in acidic urine and when exposed to light; therefore exposure to light or delay in testing can cause a false-negative result.
The reagent strip method can detect urobilinogen in concentrations as low as 0.1 mg/dL (0.1 Ehrlich unit [EU]). The reference range for urine urobilinogen by the reagent strip method is 0.0 to 1.0 mg/dL (or 0.0 to 1.0 EU/dL).
Its absence may indicate a renal disease or biliary obstruction and it is usually increased in any condition that causes an increase in the production and retention of bilirubin and in hepatitis, cirrhosis or biliary disease.
Gram-negative bacteria produce enzymes that convert urinary nitrate, a normal urine constituent, to nitrite. Nitrite reacts with chemicals in the nitrite reagent pad to form a pink color.
A positive nitrite test is an indication of the possible bacterial urinary tract infection. Examples of bacteria that frequently cause UTI and cause a positive nitrite test are gram-negative Escherichia coli, Klebsiella, Proteus, and Pseudomonas. Because certain bacteria are not capable of converting nitrates to nitrite, a negative nitrite result is possible in some UTIs. Normal urine is negative for nitrite by the reagent strip method.
Granular leukocytes, primarily neutrophils, contain an enzyme called leukocyte esterase. The presence of this enzyme in urine indicates the presence of leukocytes in urine, usually resulting from infection or inflammation in the urinary tract.
The esterase enzyme reacts with esterase substrate in the leukocyte esterase reagent pad to form a purple color. The color intensity is proportional to the number of leukocytes present.
Normal urine is negative for leukocyte esterase by the reagent strip method.
The presence of leukocytes in urine indicates an infection or inflammation. The normal result is negative.
Pyuria refers to leukocytes in urine.
Cystitis is a bladder infection while pyelonephritis is a kidney infection
The specific gravity (sp. gr.) of urine reflects the kidneys’ ability to concentrate urine. The specific gravity reagent pad contains an indicator that changes color from blue-green to green to yellow-green depending on the urine ion concentration.
Specific gravities between 1.000 and 1.030 can be measured.
The reagent strip method is not normally affected by the presence of organic molecules such as glucose, so the reagent strip sp. gr. measurement can be lower than the refractometer measurement in specimens with a positive glucose result.
Reagent strip sp. gr. can be affected by pH; neutral or alkaline urines can have falsely decreased sp. gr. by reagent strip and should be checked by another method.
The reference range of urine specific gravity in normally hydrated adults is 1.010 to 1.025 by reagent strip method.
Other components of chemical analysis are;
These are normal constituents of urine. the urinary sulfate is mainly derived from sulfur-containing amino acids and therefore determined by protein intake.
Phosphates are a normal constituent of urine also. They are important in buffering hydrogen ions in the collecting duct.
Just like the phosphates, chlorides are also normal constituents of urine. They are the major extracellular anion. It's main function is to maintain electrical neutrality, mostly as a counter-ion to sodium. It often accompanies sodium losses and excesses.
Urea. This is the end product of protein breakdown.
Uric acid is a metabolite of purine breakdown.
Creatinine is associated with muscle metabolism of creatinine phosphate.
Reference Values by Reagent Strip in urinalysis
Normal urine, when tested with a reagent strip, is negative for glucose, ketone, bilirubin, bacteria (nitrite), leukocyte esterase, and blood.
Normal urine can be negative for protein or contain a trace of protein. Normal recently voided urine
has a pH between 5.0 and 8.0 and a specific gravity between 1.010 and 1.025.
Positive or abnormal results should be confirmed according to laboratory policy.
Some test results are confirmed by retesting with a single analyte strip or a different brand of the strip. Other results must be confirmed using non-reagent strip confirmatory tests.
Positive leukocyte esterase or nitrite tests should be confirmed by microscopic examination of urine sediment.
Since urinalysis is rarely ordered as STAT tests, most laboratories have few critical urine values.