copyright © 2014. f.a. davis company chapter 5 chemical examination of urine

Copyright © 2014. F.A. Davis Company

CHAPTER 5CHAPTER 5

CHEMICAL EXAMINATION CHEMICAL EXAMINATION OF URINEOF URINE


Upon completing this chapter, the reader will be able to

1.Describe the proper technique for performing reagent strip testing.2.List four causes of premature deterioration of reagent strips, and describe how to avoid them.3.List five quality-control procedures routinely performed with reagent strip testing.4.List the reasons for measuring urinary pH, and discuss their clinical applications.5.Discuss the principle of pH testing by reagent strip.6.Differentiate between prerenal, renal, and postrenal proteinuria, and give clinical examples of each.

Learning ObjectivesLearning Objectives


7. Explain the “protein error of indicators,” and list any sources of interference that may occur with this method of protein testing.

8. Discuss microalbuminuria including significance, reagent strip tests, and their principles.

9. 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.

10.Describe the principle of the glucose oxidase method of reagent strip testing for glucose, and name possible causes of interference with this method.

11.Describe the copper reduction method for detection of urinary reducing substances, and discuss the current use of this procedure.

12.Name the three “ketone bodies” appearing in urine and three causes of ketonuria.

Learning Objectives Learning Objectives (cont’d)(cont’d)


13.Discuss the principle of the sodium nitroprusside reaction to detect ketones, including sensitivity and possible causes of interference.

14.Differentiate between hematuria, hemoglobinuria, and myoglobinuria with regard to the appearance of urine and serum and clinical significance.

15.Describe the chemical principle of the reagent strip method for blood testing, and list possible causes of interference.

16.Outline the steps in the degradation of hemoglobin to bilirubin, urobilinogen, and finally urobilin.

17.Describe the relationship of urinary bilirubin and urobilinogen to the diagnosis of bile duct obstruction, liver disease, and hemolytic disorders.

18.Discuss the principle of the reagent strip test for urinary bilirubin, including possible sources of error.



19.State two reasons for increased urine urobilinogen and one reason for a decreased urine urobilinogen.

20.Discuss the principle of the nitrite-reagent-strip test for bacteriuria.21.List five possible causes of a false-negative result in the reagent strip test for

nitrite.22.State the principle of the reagent strip test for leukocytes.23.Discuss the advantages and sources of error of the reagent strip test for

leukocytes.24.Explain the principle of the chemical test for specific gravity.25.Compare reagent strip testing for urine specific gravity with osmolality and

refractometer testing.26.Correlate physical and chemical urinalysis results.



• 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 StripsReagent Strips


• 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 Strips Reagent Strips (cont’d)(cont’d)


• 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

Reagent Strip TechniqueReagent Strip Technique


• 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 ErrorsImproper Technique Errors


• 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

Improper Technique Errors Improper Technique Errors (cont’d)(cont’d)


• 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

Handling and Storing Handling and Storing Reagent StripsReagent 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 StripsQuality Control of Reagent Strips


• 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

Quality Control of Reagent Strips Quality Control of Reagent Strips (cont’d)(cont’d)


• 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

Confirmatory TestingConfirmatory Testing


• 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 pHUrine pH


• 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

Urine pH Urine pH (cont’d)(cont’d)


• 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

Summary of Clinical Significance Summary of Clinical Significance of Urine pHof Urine pH


• 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

pH-Reagent Strip ReactionspH-Reagent Strip Reactions


• 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)

ProteinProtein


• Presence requires determination of normal or pathological condition

• Clinical proteinuria = 30 mg/dL, 300 mg/24 h• Variety of causes

– Prerenal– Renal– Postrenal

Clinical SignificanceClinical Significance


• 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)

Prerenal ProteinuriaPrerenal Proteinuria


• Multiple myeloma (plasma cell myeloma)• Immunoglobulin light chains• Multiple myeloma confirmation is serum

electrophoresis

Bence Jones Protein (BJP)Bence Jones Protein (BJP)


• Glomerular or tubular damage– Glomerular proteinuria– Microalbuminuria– Orthostatic (postural) proteinuria– Tubular proteinuria

Renal ProteinuriaRenal 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 ProteinuriaGlomerular Proteinuria


• Increased pressure on the filtration mechanism– Hypertension– Strenuous exercise– Dehydration– Pregnancy

• Preeclampsia• Benign proteinuria (transient)

– Strenuous exercise, high fever, dehydration, and exposure to cold

Glomerular Proteinuria Glomerular Proteinuria (cont’d)(cont’d)


• 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

MicroalbuminuriaMicroalbuminuria


• 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

Orthostatic (Postural) ProteinuriaOrthostatic (Postural) 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

Tubular ProteinuriaTubular 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

Postrenal ProteinuriaPostrenal Proteinuria


• 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

Protein-Reagent Strip ReactionsProtein-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 ReactionsReagent Strip Reactions


pH 3.0Indicator (H+) + Protein → Protein + H+

(Yellow) → Indicator – H+

(Green/Blue)

Reagent Strip Reactions Reagent Strip Reactions (cont’d)(cont’d)


• 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

Reaction InterferenceReaction Interference


• 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

Sulfosalicylic Acid (SSA) Sulfosalicylic Acid (SSA) PrecipitationPrecipitation


• 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

MicroalbuminuriaMicroalbuminuria


• 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

Micral-TestMicral-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

Immunodip TestImmunodip Test


• 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

Reagent Strip Microalbumin TestsReagent Strip Microalbumin Tests


• 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



• 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 StripCreatinine Reagent Strip


CuSO4 + CRE → Cu(CRE) peroxidase

Cu(CRE) peroxidase

DBDH + TMB → oxidized TMB + H2O (peroxidase) (chromogen) (orange to blue)

Creatinine Reagent Strip Creatinine Reagent Strip (cont’d)(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

Creatinine Reagent Strip Creatinine Reagent Strip (cont’d)(cont’d)


• 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): Albumin (Protein): Creatinine RatioCreatinine Ratio


• 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): Albumin (Protein): Creatinine Ratio (cont'd)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

Albumin (Protein): Albumin (Protein): Creatinine Ratio (cont'd)Creatinine Ratio (cont'd)


• The most frequent chemical analysis performed on urine• Blood and urine glucose tests are included in all physical

examinations • Mass health screening programs

GlucoseGlucose


• 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

Glucose Glucose (cont’d)(cont’d)


• 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



• 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

Nondiabetic GlycosuriaNondiabetic Glycosuria


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

pregnancy

Renal GlycosuriaRenal Glycosuria


• 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 oxidaseGlucose + O2 (air) → gluconic acid + H2O2

PeroxidaseH2O2 + chromogen → oxidized colored

chromogen + H2O



• Chromogens used– 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%

Reagent StripReagent Strip


• 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



• 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 (Clinitest)Copper Reduction Test (Clinitest)


HeatCuSO4 (cupric sulfide) + reducing substance -----

Alkali Cu2O (cuprous oxide) + oxidized substance → color

(blue/green to orange/red)

Copper Reduction TestCopper Reduction Test


• 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

Clinitest ProcedureClinitest Procedure


• 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

Reducing SubstancesReducing Substances


• 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

KetonesKetones


• 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



• Ketonuria unrelated to diabetes– Inadequate intake/absorption of carbohydrates– Vomiting– Weight loss– Eating disorders– Frequent strenuous exercise

Clinical Significance Clinical Significance (cont’d)(cont’d)


• 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

Reaction Interference Reaction Interference


• Not a urine confirmatory test• Tablet = sodium nitroprusside, glycine, disodium

phosphate, lactose (gives better color)

AcetestAcetest


• 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

BloodBlood


• Hematuria: intact RBCs, cloudy red urine• Damage to renal system

– Renal calculi– Glomerular disease– Tumors– Trauma– Pyelonephritis– Exposure to toxic chemicals– Anticoagulants

Blood Blood (cont’d)(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



• 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



• 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

BilirubinBilirubin


• 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 Bilirubin (cont’d)(cont’d)


Bilirubin MetabolismBilirubin 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



acidbilirubin 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



• 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

IctotestIctotest


• 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

UrobilinogenUrobilinogen


• 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



• 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

Clinical Significance Clinical Significance (cont’d)(cont’d)


Urine Bilirubin and Urobilinogen Urine Bilirubin and Urobilinogen in Jaundicein Jaundice

Urine Bilirubin Urobilinogen

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: acid

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



• 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)

NitriteNitrite


• 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 ReactionReagent Strip Reaction


Acidpara-arsanilic acid or sulfanilamide + NO2 —————→ diazonium salt

(nitrite)

Aciddiazonium salt + tetrahydrobenzoquinolin —————→ pink azodye

Reagent Strip Reaction Reagent Strip Reaction (cont’d)(cont’d)


• 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



• 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

Reaction Interference Reaction Interference (cont’d)(cont’d)


• 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

Leukocyte Esterase (LE)Leukocyte Esterase (LE)


• 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



• 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)

Specific GravitySpecific Gravity


Reagent Strip-Specific Gravity Reagent Strip-Specific Gravity ReactionReaction


• 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 0.005 to the reading; readers automatically add this


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