CHEMICAL EXAMINATION OF URINE

CHEMICAL EXAMINATION OF URINE
CHAPTER 5

Learning Objectives Upon completing this chapter, the reader will be able to Describe the proper technique for performing reagent strip testing. List four causes of premature deterioration of reagent strips, and describe how to avoid them. List five quality-control procedures routinely performed with reagent strip testing. List the reasons for measuring urinary pH, and discuss their clinical applications. Discuss the principle of pH testing by reagent strip. Differentiate between prerenal, renal, and postrenal proteinuria, and give clinical examples of each.

Learning Objectives (cont’d)
Explain the “protein error of indicators,” and list any sources of interference that may occur with this method of protein testing. Discuss microalbuminuria including significance, reagent strip tests, and their principles. Explain why glucose that is normally reabsorbed in the proximal convoluted tubule may appear in the urine, and state the renal threshold levels for glucose. Describe the principle of the glucose oxidase method of reagent strip testing for glucose, and name possible causes of interference with this method. Describe the copper reduction method for detection of urinary reducing substances, and discuss the current use of this procedure. Name the three “ketone bodies” appearing in urine and three causes of ketonuria.

Discuss the principle of the sodium nitroprusside reaction to detect ketones, including sensitivity and possible causes of interference. Differentiate between hematuria, hemoglobinuria, and myoglobinuria with regard to the appearance of urine and serum and clinical significance. Describe the chemical principle of the reagent strip method for blood testing, and list possible causes of interference. Outline the steps in the degradation of hemoglobin to bilirubin, urobilinogen, and finally urobilin. Describe the relationship of urinary bilirubin and urobilinogen to the diagnosis of bile duct obstruction, liver disease, and hemolytic disorders. Discuss the principle of the reagent strip test for urinary bilirubin, including possible sources of error.

State two reasons for increased urine urobilinogen and one reason for a decreased urine urobilinogen. Discuss the principle of the nitrite-reagent-strip test for bacteriuria. List five possible causes of a false-negative result in the reagent strip test for nitrite. State the principle of the reagent strip test for leukocytes. Discuss the advantages and sources of error of the reagent strip test for leukocytes. Explain the principle of the chemical test for specific gravity. Compare reagent strip testing for urine specific gravity with osmolality and refractometer testing. Correlate physical and chemical urinalysis results.

Reagent Strips Reagent strips provide a simple, rapid means for performing routine chemical tests on urine Single and multitest strips available The brand and number of tests used are a matter of laboratory preference Specified by urinalysis instrumentation manufacturers Strips consist of chemical-impregnated absorbent pads on a plastic strip

Reagent Strips (cont’d)
A color-producing chemical reaction takes place when the absorbent pad comes in contact with urine The reactions are interpreted by comparing the color produced on the pad within the required time frame with a chart supplied by the manufacturer Color comparison charts are supplied by the manufacturer Several degrees of color are shown to provide semiquantitative readings of neg, trace, 1+, 2+, 3+, and 4+ Estimates of mg/dL are also provided for many of the test areas

Reagent Strip Technique
Dip strip briefly into well-mixed specimen at room temperature Remove excess urine by touching edge of strip to container as strip is withdrawn Blot edge of strip on absorbent pad Wait specified amount of time Read using a good light source

Improper Technique Errors
Formed elements such as red and white blood cells sink to the bottom of the specimen and will be undetected in an unmixed specimen Allowing the strip to remain in the urine for an extended period may cause leaching of reagents from the pads Excess urine remaining on the strip after its removal from the specimen can produce a runover between chemicals on adjacent pads, producing distortion of the colors

Improper Technique Errors (cont’d)
The timing for reactions to take place varies between tests and manufacturers; the manufacturer’s stated time should be followed A good light source is essential for accurate interpretation of color reactions The strip must be held close to the color chart without actually being placed on the chart; reagent strips and color charts from different manufacturers are not interchangeable Specimens that have been refrigerated must be allowed to return to room temperature prior to reagent strip testing

Handling and Storing Reagent Strips
Store with desiccant in an opaque, tightly sealed container Remove strips immediately prior to use Do not expose to volatile fumes Store below 30°C Do not use past the expiration date Visually inspect for discoloration/deterioration

Quality Control of Reagent Strips
Run positive and negative controls at least once per 24 hours Run additional controls When a new bottle of strips is opened When results are questionable When there are concerns over strip integrity Record control results Manufactured positive and negative controls are available Do not use distilled water as a negative control as reactions are designed for urine ionic concentration

Quality Control of Reagent Strips (cont’d)
All negative control readings should be negative Positive control readings should agree with published control values Be aware of manufacturer-stated limitations and interfering substances Correlate chemical readings to each other and physical and microscopic readings

Confirmatory Testing Confirmatory tests use different reagents or methodologies to detect the same substances as reagent strips with the same or greater sensitivity or specificity Nonreagent strip testing procedures using tablets and liquid chemicals may be available when questionable results are obtained Chemical reliability of these procedures also must be checked using positive and negative controls

Urine pH Lungs and kidneys are major regulators of acid-base content
First morning specimen slightly acidic at 5.0 to 6.0 Postprandial specimen more alkaline Normal range is 4.5 to 8.0 No absolute values are assigned

Urine pH (cont’d) Considerations include
Acid-base content of the blood Patient’s renal function Presence of a urinary tract infection Patient’s dietary intake Age of the specimen A pH above 8.5 is associated with an aged/improperly preserved specimen, so a fresh specimen should be obtained

Summary of Clinical Significance of Urine pH
Respiratory or metabolic acidosis/ketosis Respiratory or metabolic alkalosis Defects in renal tubular secretion and reabsorption of acids and bases—renal tubular acidosis Renal calculi formation Treatment of urinary tract infections Precipitation/identification of crystals Determination of unsatisfactory specimens

pH-Reagent Strip Reactions
Needed to measure between 5.0 and 9.0 in one half or one unit increments Double-indicator system reaction Methyl red = 4 to 6 red/orange to yellow Bromthymol blue = 6 to 9 green to blue Methyl red + H+ → Bromthymol blue − H+ (Red/Orange → Yellow) (Green → Blue) Interference No known substances interfere with urinary pH measurements performed by reagent strips

Protein Most indicative of renal disease
Proteinuria seen in early renal disease Normal = <10 mg/dL or 100 mg/24 h Low-molecular-weight serum proteins are filtered; many are reabsorbed Albumin is primary protein of concern Other proteins include Vaginal, prostatic, and seminal proteins Tamm-Horsfall (uromodulin)

Clinical Significance
Presence requires determination of normal or pathological condition Clinical proteinuria = 30 mg/dL, 300 mg/24 h Variety of causes Prerenal Renal Postrenal

Prerenal Proteinuria Conditions affecting the plasma, not the kidney
Transient, increase levels of low-molecular-weight plasma proteins, acute phase reactants, exceed reabsorptive capacity Rarely seen on reagent strip (not albumin)

Bence Jones Protein (BJP)
Multiple myeloma (plasma cell myeloma) Immunoglobulin light chains Multiple myeloma confirmation is serum electrophoresis

Renal Proteinuria Glomerular or tubular damage Glomerular proteinuria
Microalbuminuria Orthostatic (postural) proteinuria Tubular proteinuria

Glomerular Proteinuria
Damage to glomerular membrane Impaired selective filtration causes increased protein filtration leading to cellular excretion Abnormal substances deposit on the membrane Primarily immune disorders result in immune complex formation Lupus erythematosus, streptococcal glomerulonephritis Amyloids and other toxins

Glomerular Proteinuria (cont’d)
Increased pressure on the filtration mechanism Hypertension Strenuous exercise Dehydration Pregnancy Preeclampsia Benign proteinuria (transient) Strenuous exercise, high fever, dehydration, and exposure to cold

Microalbuminuria Diabetic nephropathy with type 1 and type 2 diabetes mellitus Reduced glomerular filtration Eventual renal failure Also associated with an increased risk of cardiovascular disease

Orthostatic (Postural) Proteinuria
Increased pressure on the renal vein when in the vertical position Occurs in vertical position, disappears in horizontal position Collection instructions Empty bladder before bed Collect specimen immediately on arising Negative reading will be seen on the first morning specimen Positive result will be found on the second specimen

Tubular Proteinuria Tubular damage affecting reabsorptive ability
Acute tubular necrosis Toxic substances, heavy metals, viral infections, Fanconi syndrome (generalized proximal convoluted tubule defect) Amount of protein Glomerular disorders: up to 4 g/day Tubular disorders: much lower levels

Postrenal Proteinuria
Protein added in the lower urinary and genitourinary tract Microbial infections causing inflammations and release of interstitial fluid protein Menstrual contamination Semen/prostatic fluid Vaginal secretions Traumatic injury

Protein-Reagent Strip Reactions
Traditional principle Protein error of indicators Certain indicators change color in the presence of protein at a constant pH Protein accepts H+ from the indicator, increased sensitivity to albumin due to more amino groups to accept H+ than other proteins

Reagent Strip Reactions
Tetrabromophenol blue or tetrachlorophenol tetrabromosulfonephthalein and an acid buffer pH level 3 both indicators are yellow Color progresses through green to blue Report: neg, trace, 1+, 2+, 3+, 4+, or 30, 100, 300, 2000 mg/dL Trace values are <30 mg/dL

Reagent Strip Reactions (cont’d)
pH 3.0 Indicator (H+) + Protein → Protein + H+ (Yellow) → Indicator – H+ (Green/Blue)

Reaction Interference
Highly buffered alkaline urine overrides acid buffer system (color change unrelated to protein concentration) Leaving reagent pad in urine too long removes buffer False-positive Highly pigmented urine High SG Quaternary ammonium compounds, detergents, antiseptics, chlorhexidine

Sulfosalicylic Acid (SSA) Precipitation
Confirmatory test for protein Cold precipitation test that reacts equally with all forms of protein Must be performed on centrifuged specimens to remove any extraneous contamination

Microalbuminuria Semiquantitative testing for patients at risk for renal disease Immunochemical assays for albumin or albumin-specific reagent strips Measure creatinine to produce an albumin:creatinine ratio First morning specimens are recommended

Micral-Test Gold-labeled antihuman antibody-enzyme conjugate
Dip strip in urine to marked level for 5 seconds Albumin binds to antibody Bound and unbound conjugates move up strip Unbound removed in captive zone containing albumin; bound continues up strip Reaches enzyme substrate, reacts Colors from white (neg) to red (varying degrees) Compare color to chart Results read from 0 to 10 mg/dL

Immunodip Test Immunochromographic technique
Specially designed container for strip Place container in controlled amount of specimen for 3 min, urine enters container Albumin binds to blue latex particles coated with antihuman albumin antibody Bound and unbound migrate up strip Unbound encounters area of immobilized albumin on strip—forms blue band Bound continues migrating to an area of immobilized antibody and forms blue band Color of band is compared with chart

Reagent Strip Microalbumin Tests
Clinitek microalbumin reagent strips and Multistix Pro reagent strips Simultaneous measurement of albumin and creatinine Provide an estimate of the 24-hour albumin concentrations from random urine Albumin pad uses dye-binding reaction for specific albumin testing

Albumin strip dye (DIDNTB) bis(3′,3″, diodo-4′,4″-dihydroxy-5′,5″-dinitrophenyl)-3,4,5,6-tetra-bromo-sulfonphthalein Specific for albumin Sensitivity: 8 to 15 mg/dL (80 to 150 mg/L) Highly buffered alkaline urine interference is controlled by treated paper Polymethyl vinyl glycol carbonate decreases nonspecific binding of poly amino acids Visibly bloody urine elevates results Abnormally colored urines may interfere with readings

Creatinine Reagent Strip
Principle: pseudoperoxidase activity of copper-creatinine complexes Reagent strips contain copper sulfate (CuSO4, 3,3′,5,5′-tetramethylbenzidine (TMB) and diisopropyl benzene dihydroperoxide (DBDH)) Creatinine in urine combines with copper sulfate to form copper-creatinine peroxidase Peroxidase reacts with DBDH, releases oxygen ions that oxidize TMB Colors change from orange to green to blue

Creatinine Reagent Strip (cont’d)
CuSO4 + CRE → Cu(CRE) peroxidase Cu(CRE) peroxidase DBDH + TMB → oxidized TMB + H2O (peroxidase) (chromogen) (orange to blue)

Creatinine Reagent Strip (cont’d)
Results: 10, 50, 100, 200, 300 mg/dL or 0.9, 4.4, 8.8, 17.7, 26.5 mmol/L Elevated results: bloody urine, tagamet (cimetidine), abnormal urine color No creatinine results are abnormal Purpose is to correlate creatinine with albumin results to determine the albumin:creatinine ratio

Albumin (Protein): Creatinine Ratio
Automated and manual methods available Clinitek microalbumin strips can be read only on Clinitek instruments Instrument calculates A:C ratio and prints out albumin, creatinine, and A:C results Results in conventional and SI units Abnormal A:C ratio: 30 to 300 mg/g or 3.4 to 33.9 mg/mol

Albumin (Protein): Creatinine Ratio (cont'd)
Bayer Multistix Pro 10 strips measure creatinine, protein-high and protein-low Protein-high is protein error of indicators method Protein-low is dye-binding method Urobilinogen and bilirubin are not included on these strips Read manually or on instrumentation Print-out is protein:creatinine ratio with albumin result included on print-out

Albumin (Protein): Creatinine Ratio (cont'd)
Instrument automatically calculates A chart is available for manual ratio calculation Results are reported as Normal or Abnormal A result of normal dilute indicates that the specimen should be recollected, making sure it is a first morning specimen

Glucose The most frequent chemical analysis performed on urine
Blood and urine glucose tests are included in all physical examinations Mass health screening programs

Glucose (cont’d) Clinical significance
Major screening test for diabetes mellitus Renal threshold is 160 to 180 mg/dL Higher blood sugar = glycosuria Gestational diabetes Placental hormones block action of insulin High fetal glucose stresses baby’s pancreas Result is fat baby Mother prone to type 2 diabetes

Elevated blood glucose, diabetes mellitus Renal threshold is ~160 to 180 mg/dL Higher blood sugar = glycosuria Collection under controlled conditions Fasting specimen “Second” collection 2 h postprandial

Nondiabetic Glycosuria
Hormonal disorders: pancreatitis, pancreatic cancer, acromegaly, Cushing’s syndrome, hyperthyroidism, pheochromocytoma Hormones: glucagon, epinephrine, cortisol, thyroxine, growth hormone oppose glucose Insulin: converts glucose to storage glycogen Hormones: glycogen back to glucose Epinephrine: inhibits insulin; seen with stress, cerebral trauma, and myocardial infarction

Renal Glycosuria Tubular reabsorption disorder End-stage renal disease
Cystinosis Fanconi syndrome Temporary lowering of renal threshold in pregnancy

Glucose oxidase reaction specific for glucose Glucose oxidase, peroxide, chromogen, buffer on test pad Double sequential enzyme reaction Glucose oxidase catalyzes a reaction between glucose and oxygen Produces gluconic acid and peroxide Peroxidase catalyzes the reaction between peroxide and chromogen to form an oxidized colored compound Direct proportion to the concentration of glucose

Glucose oxidase Glucose + O2 (air) → gluconic acid + H2O2 Peroxidase H2O2 + chromogen → oxidized colored chromogen + H2O

Reagent Strip Chromogens used Reporting results
Potassium iodide (green to brown) (Multistix) Tetramethylbenzidine (yellow to green) (Chemstrip) Reporting results Neg, trace, 1+, 2+, 3+, 4+ 100 mg/dL to 2 g/dL 0.1% to 2%

False-positive: only peroxide, oxidizing detergents False-negative: enzymatic reaction interference Ascorbic acid and strong reducing agents High levels of ketones (unlikely) High specific gravity and low temperature Greatest source of error is old specimens Subjecting the glucose to bacterial degradation

Copper Reduction Test (Clinitest)
Reduction of copper sulfate to cuprous oxide with alkali and heat Clinitest tablets: copper sulfate, sodium carbonate, sodium citrate, sodium hydroxide Sodium citrate + NaOH = heat Sodium carbonate = CO2 blocks room air Reducing substance + CuSO4 Color change: negative blue (CuSO4) through green, yellow, and orange/red (Cu2O)

Copper Reduction Test Heat Alkali
CuSO4 (cupric sulfide) + reducing substance ----- Alkali Cu2O (cuprous oxide) + oxidized substance → color (blue/green to orange/red)

Clinitest Procedure Pass through
High levels of reducing substance Color from blue through red back to green-brown: rapid reaction Repeat with two-drop procedure 10 drops water 2 drops urine Values up to 5 g/L versus 2 g/L Separate chart must be used Hygroscopic tablets: strong blue color and excess fizzing = deterioration

Reducing Substances Not a specific test for glucose
Sensitivity: 200 mg/dL (lower) than strip Clinitest does not provide a confirmatory test for glucose Interference from reducing sugars Galactose, lactose, fructose, maltose, pentoses, ascorbic acid, cephalosporins Major use is quick screen for “inborn error of metabolism” in children up to 2 years old Newborn screening programs for galactosemia in all states

Ketones Three intermediate products of fat metabolism
Acetone: 2% Acetoacetic acid: 20% β-hydroxybutyrate: 78% Appear in urine when body stores of fat must be metabolized to supply energy

Increased fat metabolism = inability to metabolize carbohydrate Primary causes Diabetes mellitus Vomiting (loss of carbohydrates) Starvation, malabsorption, dieting (↓ intake) Ketonuria shows insulin deficiency Monitor diabetes Diabetic ketoacidosis = increased accumulation of ketones in the blood Electrolyte imbalance, dehydration, and diabetic coma

Clinical Significance (cont’d)
Ketonuria unrelated to diabetes Inadequate intake/absorption of carbohydrates Vomiting Weight loss Eating disorders Frequent strenuous exercise

Primary reagent: sodium nitroprusside (Nitroferricyanide) Measure primarily acetoacetic acid Assumes the presence of β-hydroxybutyrate and acetone Acetoacetic acid (alkaline) + nitroprusside → purple color

Report qualitatively Negative Trace Small (1+) Moderate (2+) Large (3+) Semiquantitatively Negative Trace (5 mg/dL) Small (15 mg/dL) Moderate (40 mg/dL) Large (80 to 160 mg/dL)

acetoacetate (and acetone) + sodium nitroprusside Alkaline + (glycine) ——————> purple color

Levodopa in large dosage Medications containing sulfhydryl groups May produce atypical color reactions False-positive results from improperly timed readings Falsely decreased values in improperly preserved specimens Breakdown of acetoacetic acid by bacteria

Acetest Not a urine confirmatory test
Tablet = sodium nitroprusside, glycine, disodium phosphate, lactose (gives better color)

Blood Hematuria: intact RBCs
Cloudy red urine Hemoglobinuria: product of RBC destruction Clear red urine Any amount of blood greater than five cells per microliter of urine is considered clinically significant Chemical tests for hemoglobin provide the most accurate means for determining the presence of blood The microscopic examination can be used to differentiate between hematuria and hemoglobinuria

Blood (cont’d) Hematuria: intact RBCs, cloudy red urine
Damage to renal system Renal calculi Glomerular disease Tumors Trauma Pyelonephritis Exposure to toxic chemicals Anticoagulants

Blood (cont’d) Hemoglobinuria: clear, red urine
Transfusion reactions Hemolytic anemias Severe burns Infections/malaria Strenuous exercise/RBC trauma Brown recluse spider bites Hemoglobinuria may result from the lysis of red blood in dilute, alkaline urine Hemosiderin: yellow brown granules in sediment

Blood (cont’d) Myoglobinuria: heme-containing protein in muscle tissue; clear, red/brown urine Rhabdomyolysis: muscle destruction Muscular trauma/crush syndromes Prolonged coma Convulsions Muscle-wasting diseases Alcoholism Drug abuse Extensive exertion Cholesterol-lowering statin medications

Principle pseudoperoxidase activity of hemoglobin Catalyze a reaction between the heme component Hemoglobin and myoglobin Chromogen tetramethylbenzidine Produce an oxidized chromogen Green-blue color

hemoglobin H2O2 + chromogen  oxidized chromogen + H2O peroxidase Two charts corresponding to different reactions Free hemoglobin shows uniform color Intact RBCs show a speckled pattern on pad Report: trace, small (1+), moderate (2+), large (3+) Sensitivity 5 RBCs/μL

False-positive Menstrual contamination, strong oxidizing agents, bacterial peroxidases False-negative Ascorbic acid >25 mg/dL High SG/crenated cells Formalin Captopril High concentrations of nitrite Unmixed specimens

Bilirubin Urine bilirubin early indicator of liver disease
Normal degradation product of hemoglobin RBCs destroyed by liver and spleen following 120-day life span Body recycles iron, protein Protoporphyrin is broken down into bilirubin Bilirubin is bound to albumin Kidneys cannot excrete Unconjugated bilirubin: water insoluble

Bilirubin (cont’d) Conjugated bilirubin: water soluble
Unconjugated bilirubin to the liver Conjugated with glucuronic acid Forms conjugated bilirubin From liver to intestines Reduced to urobilinogen, stercobilinogen, and urobilin by intestinal bacteria Excreted in feces

Bilirubin Metabolism

Conjugated bilirubin appears in urine with bile duct obstruction, liver disease or damage Obstruction: bilirubin backs up into circulation and is excreted in urine No urobilinogen is formed Hepatitis, cirrhosis: conjugated bilirubin leaks back into circulation from damaged liver; some bilirubin passes to intestine Hemolytic disease: increased unconjugated bilirubin, increased urobilinogen

Principle is a diazo reaction Report: neg, small (1+), moderate (2+), large (3+) Colors may be difficult to interpret Easily influenced by other pigments present in the urine Atypical colors can be problem for automated readers

acid bilirubin glucuronide + *diazonium salt  azodye (tan or pink to violet) * diazonium salt- (2,4-dichloroaniline diazonium salt or 2,6-dichlorobenzene-diazonium-tetrafluoroborate)

False-positive Urine pigments Pyridium (phenazopyridine) Drugs indican, iodine False-negative Old specimens (biliverdin does not react) Ascorbic acid >25 mg/dL Nitrite Combine with diazonium salt and block bilirubin reaction

Ictotest Confirmatory for bilirubin
Tablets containing p-nitrobenzene-diazonium-p-toluenesulfonate, SSA, sodium carbonate, and boric acid Use specified mat for test; mat keeps bilirubin on surface for reaction Positive reaction = blue-to-purple color Interfering substances are washed into the mat, and only bilirubin remains on the surface

Urobilinogen Bilirubin in intestine converted to urobilinogen and stercobilinogen Urobilinogen is reabsorbed into circulation and stercobilinogen is not = urobilin Pigments responsible for the characteristic brown color of feces There is always a small amount of urobilinogen filtered by the kidneys and is found in the urine <1 mg/dL

Early detection of liver disease, greater than 1 mg/dL Liver disorders, hepatitis, cirrhosis, carcinoma Hemolytic disorders Excess bilirubin being converted to urobilinogen and ↑ urobilinogen recirculated to liver Negative bilirubin and strong positive urobilinogen are seen in hemolytic disorders

Clinical Significance (cont’d)
1% of the nonhospitalized population and 9% of a hospitalized population exhibit elevated results This is frequently caused by constipation No urobilinogen is seen in the urine with bile duct obstruction; strip will give a normal result Reagent strips cannot report a negative urobilinogen reading

Urine Bilirubin and Urobilinogen in Jaundice
Bile duct obstruction + + + Normal Liver damage + or − ++ Hemolytic disease Negative

Different principles for Multistix and Chemstrip Multistix: Ehrlich’s aldehyde reaction p-dimethylaminobenzaldehyde (Ehrlich reagent); report in Ehrlich units (EU) 1 EU = 1 mg/dL Normal readings 0.2 to 1, abnormal 2, 4, 8 Light to dark pink Chemstrip: diazo (azo-coupling) reaction 4-Methoxybenzene-diazonium-tetrafluoroborate; more specific than Ehrlich reaction; report in mg/dL White to pink

MULTISTIX: acid urobilinogen + p-dimethylaminobenzaldehyde → red color * ERC (Ehrlich’s reagent) Chemstrip: urobilinogen + **diazonium salt ———-----→ red azodye *(Ehrlich reactive compounds) **(4-methyloxybenzene-diazonium-tetrafluoroborate)

Ehrlich reactive compounds: porphobilinogen, indican, sulfonamides, methyldopa, procaine, chlorpromazine, p-aminosalicylic acid Both tests: urobilinogen is highest after meals (increased bile salts), old specimens and formalin preservation decrease results Chemstrip: false-negative with high nitrite interferes with diazo reaction

Nitrite Clinical significance
Rapid screening test for the presence of urinary tract infection (UTI) Cystitis (initial bladder infection) Pyelonephritis (tubules) Evaluation of antibiotic therapy Monitoring of patients at high risk for urinary tract infection Screening of urine culture specimens (in combination with LE test)

Reagent Strip Reaction
Tests ability of bacteria to reduce nitrate (normal constituent) to nitrite (abnormal) Greiss reaction: nitrite reacts with aromatic amine to form a diazonium salt that then reacts with tetrahydrobenzoquinoline to form a pink azodye Correspond with a quantitative bacterial culture criterion of 100,000 organisms/mL Results: negative and positive

Reagent Strip Reaction (cont’d)
Acid para-arsanilic acid or sulfanilamide + NO2 —————→ diazonium salt (nitrite) diazonium salt + tetrahydrobenzoquinolin —————→ pink azodye

False-negative Nonreductase-containing bacteria Insufficient contact time between bacteria and urinary nitrate Lack of urinary nitrate Large quantities of bacteria converting nitrite to nitrogen Presence of antibiotics High concentrations of ascorbic acid High specific gravity Negative results in the presence of even vaguely suspicious clinical symptoms should always be repeated or followed by a urine culture

Reaction Interference (cont’d)
False-positive Old specimens (bacterial multiplication) Highly pigmented urine Pink edge or spotting on reagent strip is considered negative Check automated readers manually for color interference

Leukocyte Esterase (LE)
Standardized means for the detection of leukocytes Purpose is to detect leukocytes so as not to rely on microscopic LE test detects the presence of esterase in the granulocytes and monocytes Advantage: detects presence of lysed leukocytes Not considered a quantitative test: do microscopic if positive

Bacterial and nonbacterial urinary tract infection Inflammation of the urinary tract Screening of urine culture specimens in conjunction with nitrite but a better predictor than nitrite Also seen with Trichomonas, Chlamydia, yeast, interstitial nephritis

LE catalyzes hydrolysis of acid esterase on pad to aromatic compound and acid; aromatic compound reacts with diazonium salt on pad for purple color Leukocyte indoxylcarbonic acid ester —————→ indoxyl + acid indoxyl Esterase Acid + diazonium salt ————→ purple azodye

LE reaction requires the longest time of all the reagent strip reactions 2 minutes Reported as Negative Trace Small: 1+ Moderate: 2+ Large: 3+ Trace readings may not be significant and should be repeated on a fresh specimen

False-positive Strong oxidizing agents Formalin Highly pigmented urine, nitrofurantoin False-negative High concentrations of protein, glucose, oxalic acid, ascorbic acid Crenation from high specific gravity Inaccurate timing: must have 2 min Presence of the antibiotics; gentamicin, cephalosporins, tetracyclines

Specific Gravity Based on pKa (dissociation constant) of a polyelectrolyte in alkaline medium Polyelectrolyte ionizes releasing H+ in relation to concentration of urine ↑concentration = more H+ released Indicator bromthymol blue measures pH change Blue (alkaline) through green to yellow (acid)

Reagent Strip-Specific Gravity Reaction

No interference from large molecules, glucose and urea and radiographic dye and plasma expanders Reason for difference in refractometer reading Slight elevation from protein Decreased readings: urine pH 6.5 or higher Interferes with indicator; add to the reading; readers automatically add this

CHEMICAL EXAMINATION OF URINE

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