Subtopic 2: Chemical Testing

Reagent-strip Testing

A plastic strip is used, which contains pads that have incorporated within them the reagents for chemical reactions for the detection of a number of urine constituents. Urine is added to the pads for reaction by dipping the plastic strip into the urine and then slowly withdrawing it. The subsequent colorimetric reactions are timed to an endpoint; the extent of colors formation is directly related to the level of the urine constituent. The colors can be read manually by comparison with color charts or with the use of automated reflectance meters.

The following are four general rules to be followed when performing urine reagent strip testing.

  1. Test urine promptly; use properly timed test readings only.

  2. Beware of interfering substances.

  3. Understand the advantages and limitations of the test.

  4. Employ controls at least once per day, documenting the results.

Positive results from reagent-strip testing may require confirmation with chemical and microscopic methods.

Manufacturers' information on sources of inhibitors and false-positive and false-negative results can be identified from the package inserts of the test strips. For example, ascorbic acid in urine can interfere with reagent-strip reactions for glucose, hemoglobin, bilirubin, and nitrite.

Manufacturers have been encouraged to minimize or eliminate this interference when possible because ingestion of vitamin C supplements is so common. Some reagent test strips have an additional test reaction that measures the levels of urinary ascorbic acid, and serve as a reminder of the possibility of interference from this source.

Test Principles, Significance, and Normals

  1. pH

  2. Proteins

  3. Sugars

  4. Ketones

  5. Blood and myoglobin

  6. Bilirubin

  7. Urobilinogen

  8. Nitrities

  9. Leukocyte esterase

1. pH

Most urinalysis laboratories use multi-test reagent strips containing two pH indicators, methyl red and bromthymol blue. These provide a range of sensitivity to pH from 5.0 to 9.0; the pH is reflected by a color which can change from orange (acid) to green to blue (alkaline).

Fresh urine specimens can have pH values ranging from acidic to alkaline. Upon standing the decomposition of urea into ammonia causes the urine to become more alkaline. Lower pH values are observed in cases of diabetes and in patients with fever. Urine retention by some patients can result in more alkaline urine. The standard method for pH measurements uses glass electrodes. Urinary pH measured with indicator paper is more than accurate enough for clinical purposes, since small changes in urinary pH are of little clinical significance. There is no confirmatory testing for urine pH.

Clinical significance—Common clinical causes of acidic and alkaline urine are listed in the following table. The normal American diet is high in protein that results in acidic urine (pH 5.0 to 6.5). Alkaline urines (pH 8.0 to 8.5) are more often associated with an unpreserved or old specimen, which turns alkaline as the result of urease-producing ammonia bacteria, such as Proteus species. Patients with renal tubular acidosis, a clinical syndrome characterized by an inability to excrete an acidic urine, may produce urine with a much higher pH than would be expected on the basis of the acidosis.

Normal—The urinary pH range is usually 4.7 to 7.8. Extremely acidic or alkaline urine usually indicates a poorly collected specimen.

2. Proteins

Reagent-strip test for protein is a semi-quantitative screening procedure for proteinuria. Reagent strips contain a pH sensitive dye; tetrabromphenol blue and 31, 35, 51, 55 tetrachlorophenol-3,4,5,6-tetrabromosulfophthalein. The presence of protein on the strip changes the pH environment of the dye embedded in the pad, resulting in a change in color.

  pH 3  
Tetrabromphenol blue Positive results (green-blue)

  pH 3  
Tetrabromphenol blue Negative results (yellow)
  No protein  

Strip tests are more sensitive for albumin and can detect other proteins at higher concentrations. Thus, because there is a risk for false-negative results, it is recommended that the laboratory consider simultaneously performing both a reagent-strip test and an acid precipitation test for the detection of all types of proteinuria. A faintly positive result should be confirmed with a more specific test such as the trichloroacetic acid or the sulfosalicylic acid tests. A grossly positive SSA or TCA turbidity test result may indicate the presence of drugs or Bence Jones proteins.

False positive results maybe caused by alkaline or buffered urine as well as by quarternary ammonium compounds found in detergents.

Clinical significance—Most of the urine protein is albumin, which has crossed the glomerular membrane. Smaller-molecular-weight proteins such as globulins may also be present in urine. Once filtered at the glomerulus, proteins are almost completely reabsorbed in the proximal tubule. Proteinuria, therefore, can be the result of either increased filtration at the glomerulus or decreased tubular reabsorption. Glomerular proteinuria is associated with the presence of larger molecular weight proteins and larger protein losses, usually > 2 g/day. The nephrotic syndrome is associated with very large losses of protein, usually >2-3 g/day. Tubular proteinuria is associated with smaller amounts (1-3 g/day) of lower molecular weight protein molecules. Small losses of protein in urine can be seen with vigorous exercise and pregnancy.

Measurement of urinary pH is also useful for managing patients with renal stones or crystals. Uric acid stones form in acidic urine and are more soluble in alkaline urines. However, alkaline urine will precipitate calcium or calcium phosphate crystals, while an acidic urine will tend to dissolve them. Inducing an alkaline urine during sulfonamide and streptomycin therapy is done to prevent precipitation of these drugs in the kidneys and to prevent the formation of uric acid, cystine, and oxalate stones. Alkaline urines are also desirable during treatment of transfusion reactions and salicylate intoxication. An acidic pH is used to combat bacteriuria in patients with cystitis and to prevent formation of alkaline stones. Technologists should be aware that alkaline urine interferes with the determination of proteins by the reagent strip technology and may alter the urine sediment examination.

Normal—A healthy person will excrete up to approximately 100 mg/day, a very small fraction of the plasma protein that is filtered at the glomerulus.

3. Sugars

Enzymatic testing—The reagent-strip tests are highly specific for glucose. They detect the oxidation of glucose to gluconic acid:

  Glucose oxidase  
Glucose + Oxygen in room air Gluconic acid + Hydrogen peroxide

Hydrogen peroxide + Chromogen Oxidized chromogen (blue) + H2O

Tetramethylbenzidine and o-toluidine have been used as the chromogen for the indicator reaction.

Copper reduction (Clinitest, Benedict's test)—The Clinitest tablet (Ames Division, Miles Laboratories, Inc., Elkhart, IN) usually serves as a confirmatory test for sugar. Using the principle of the reduction of cupric salts by reducing sugars; including glucose, galactose, lactose, and pentoses; the copper reduction test measures total reducing substances in urine.

Cupric ions + Glucose
(or other reducing substances)
+ Cuprous
  Alkali (red)   (yellow)

Clinical significance—Glucose is the predominant sugar in urine. It is not detectable by reagent strips in the urine of healthy individuals. Temporary elevation of glucose excretion measurable by test strips can occur after treatment with some drugs, cases of shock and during pregnancy. Repeated positive testing is almost always diagnostic for diabetes.

Reagent strips will detect glucose at a concentration of 400 to 750 mg/L (2.2 to 3.85 mmol/L), while the Clinitest will detect reducing substances at a concentration of 2000 mg/L (~ 11 mmol/L) or greater. The copper reduction method will also detect ascorbic acid and certain drugs, such as, nalidixic acid [NegGram]), used to treat urinary tract infections; probenecid, used to treat gout; and cephalosporin, an antibiotic.

Because Clinitest is both less specific and less sensitive than the reagent strip, it cannot be used as a confirmatory test for a positive reagent-strip glucose test. Clinitest should be reserved for patient populations in whom non-glucose reducing substances need to be detected, such as in newborn screening. A negative dipstick for glucose, but a positive Clinitest would suggest the presence of non-glucose sugar, requiring additional testing. This type of testing should be routine for all newborns. The presence of ascorbic acid may lead to erroneous low results.

Normal—Health individuals normally will have no detectable sugars in their urine.

4. Ketones

Ketone bodies is a term used to describe three discrete but metabolically related chemicals: acetoacetic acid, b-hydroxybutyric acid, and acetone. Reagent-strip testing for ketones uses the sodium nitroprusside (sodium nitroferricyanide) reaction, which detects acetone and acetoacetic acid but not b-hydroxybutyric acid, the primary ketone body.

  Alkaline pH  
Acetoacetic acid + Sodium nitroprusside + Glycine Purple color

Specific measurement of acetoacetic acid, b-hydroxybutyric acid, and acetone can be accomplished using enzymatic procedures.

Clinical significance—Ketones are spilled into urine when the body cannot utilize glucose (as in diabetes) and metabolize fatty acids. This catabolism is incomplete, resulting in the formation of large amounts of acetoacetic acid, acetone and beta-hydroxybutyric acid (ketone bodies).

It is important to realize that the sodium nitroprusside reagent reacts primarily with acetoacetic acid; acetone has only a 20% reactivity compared with acetoacetate, while b-hydroxybutyric acid does not react at all in this reaction. So this method will always underestimate the total load of excreted ketone bodies. However, this error is probably of no practical significance in the diagnosis or monitoring of diabetes mellitus.

Normal—Health individuals normally will have no detectable ketones in their urine.

5. Blood and Myoglobin

The reagent-strip method for hemoglobin and myoglobin uses the peroxidase-like activity of these heme-proteins to oxidize a chromogen (usually tetramethyl benzidine) to a colored product:

  Alkaline pH  
Hydrogen peroxide (H2O2)
+ Chromogen
Oxidized chromogen (blue)
+ H2O

A positive test indicates the presence of red blood cells in the urine (hematuria), free hemoglobin in the urine (hemoglobinuria), or myoglobinuria. A microscopic urinalysis should be performed to confirm the presence of intact erythrocytes.

Clinical significance—Oxidizing agents such as iodides and bromides in the urine may cause false-positive results; large quantities of ascorbic acid (used in some antibiotics) in the urine may produce false-negative results with some reagent strips. The peroxidase assay cannot distinguish between the presence of hemoglobin or myoglobin in urine. The presence of intact red blood cells would strongly suggest hematuria, but in appropriate clinical conditions, such as a crush injury, a specific test for myoglobin will need to be performed.

Normal—Health individuals normally will have no detectable blood or myoglobin in their urine.

6. Bilirubin

The reagent-strip method for determining bilirubin involves a diazotization reaction:

Bilirubin glucuronide
+ Diazonium salt
Azobilirubin (brown)

The diazonium salts 2,4-dichlorobenzenediazonium-tetrafluroborate and 2,4-dichloroaniline are both used for this test. While this reaction will occur with any form of bilirubin, only the water soluble, conjugated form is present in urine. Thus the reaction is indicative of conjugated bilirubin in urine.

Clinical significance—The pigment bilirubin is formed by the degredation of heme. It is not excreted into urine. Only the conjugated form of bilirubin, often termed direct bilirubin, is excreted into urine. In most healthy individuals the amount of conjugated bilirubin excreted is not detected by the strips. In cases when bilirubin is elevated and is conjugated, it will be detected by the test strip. A number of liver diseases such as viral hepatitis will result in elevated urine bilirubin.

A negative bilirubin result on a urine from a patient believed to have increased serum bilirubin, and a questionably positive result, such as from a highly colored urine, should be confirmed by using Ictotest tablets (Ames Division, Miles Laboratories, Inc., Elkhart, IN). However, positive bilirubin results do not need to be routinely confirmed. The Ictotest employs the same diazotization reaction as the reagent strip, but may not give a false positive result with colored urines. False-negative results may occur if the urine is not fresh, because urinary bilirubin may hydrolyze or oxidize when exposed to light.

Normal—Urine from healthy individuals does not contain detectable bilirubin.

7. Urobilinogen

The methods for detecting urinary urobilinogen differ with the manufacturer of the reagent-strip tests. Ames (Miles Division, Bayer, Inc.) employ reagent-strip tests containing p-dimethylaminobenzaldehyde which reacts in a simple color reaction with porphobilinogen, known as the Ehrlich reaction. Boehringer Mannheim Diagnostics (BMD) reagent test strip use a reaction with 4-methoxybenzene-diazonium-tetrafluroborate that is more specific for urobilinogen; urinary urobilinogen reacts with the diazonium compound to form a red color.

Clinical significance The Ehrlich reaction is not specific for urobilinogen, and false-positive findings may result from other Ehrlich reagent positive compounds (porphobilinogen, p-aminosalicylic acid) (PAS). However, the presence of compounds producing false positive results is usually not a clinical problem. It should be noted that reagent strips will not detect the absence of urinary urobilinogen, that is, the levels of urinary urobilinogen associated with decreased production of urobilinogen because of hepatic obstructive disease will not be differentiated from the lowest detectable color (2 mg/L).

A fresh specimen is essential for the detection of urobilinogen, as it is a light-sensitive compound. The preferred specimen for detecting and/or quantifying urinary urobilinogen is a 2-hour early afternoon specimen. This collection takes into account the diurnal excretion pattern of urobilinogen.

Normal—A healthy person will contain from 2 to 10 mg/L of urobilinogen.

8. Nitrites

The nitrite test is used in urinalysis laboratories to detect bacteriuria. The reagent-strip nitrite test measures the nitrite formed by the enzymatic reduction of nitrate by certain bacteria in urine. Two reactions are currently being employed. In one (Ames), nitrite reacts with p-arsanilic acid under reaction conditions of acid pH to form a diazonium compound; the diazonium product reacts with N-1-naphthyl ethylenediamine to produce a pink color. The BMD product uses 3-hydroxy-1,2,3,4 tetrahydro-7,8-benzoquinoline and sulfanilamide in a similar reaction.

Clinical significance A positive nitrite test is dependent upon the excretion of nitrate in the diet into urine where it is converted to nitrite by gram negative bacteria.

The sensitivity of the nitrite test is about 60% when compared with microbiological procedures. There are very few cases of false-positive nitrite results.

Normal Urine from healthy individuals does not contain detectable nitrite.

9. Leukocyte esterase

Reagent-strip tests for pyuria (leukocytes in urine) are based on the presence of intracellular esterases of bacteria. The esterases catalyze the hydrolysis of esters, releasing a product that reacts in a subsequent reaction with a diazonium salt to produce a colored product. The Ames product uses a derivitized pyrrole amino acid ester as the substrate for the esterases, while the BMD product employs an indoxylcarbonic acid ester. The intensity of both color reactions is proportional to the number of leukocytes in the specimen. The assay will detect both lysed and intact leukocytes. Sensitivities for the two reagent-strip manufacturers are listed in the Table of Practical Sensitivities of Two Reagent Strip Tests. False-positive results are seen with trichomonads and oxidizing agents; eosinophils and histiocytes may also produce a positive reaction. Elevated levels of urinary protein and ascorbic acid may result in false-negative values. The leukocyte test has been suggested as a screening test for pyuria; only urine specimens that are positive for leukocytes by the esterase reagent strip test, would require the more time-consuming microscopic examination for leukocytes.

Normal Urine from healthy individuals does not contain detectable leukocytes.