Chemical Examination of Urine

Chemical Examination

of UrineRicki Otten MT(ASCP)[email protected]

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Objectives:

• Review the objectives on page 1 and 2 of the lecture handout

• Objectives marked with ‘*’ will not be tested over during student lab rotation

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Historical Perspective: Urinalysis• Physical examination of urine

– Odor– Taste– Color– Clarity

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Historical Perspective• Chemical examination of urine

– Limited reactions– Required large volumes of urine– Large volumes of reagent– Performed in test tubes– Time consuming and cumbersome– Clinical usefulness was not realized– Not routinely ordered

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Historical Perspective• Microscopic examination of urine

– Not until invention of the microscope– Then clinical usefulness realized

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Reagent Strip Testing

• Technology and necessity• Chemical reactions ‘miniaturized’• Required less urine• Test results within minutes• Easy to perform• Increased test utilization

Brunzel, 2nd Ed, page 124

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• Ideal qualitative screening tool– Sensitive: Low concentration of substances

Negative result = normal

– Specific: Reacts with only one substance False negative and false positive

– Cost effective: Relatively inexpensive tool that provides information about the health statusof the patient

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Reagent Strip Testing• Chemically impregnated absorbent

pads attached to an inert plastic strip

• Each pad is a specific chemical reaction thattakes place upon contact with urine

• Chemical reaction causes the color of the pad tochange

• Color compared to a color chart for interpretation

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• Qualitative or semi-quantitative results– Concentration units (mg/dl)– Negative, small, moderate large– Negative, 1+, 2+, 3+, 4+

• Timing of chemical reactions is CRITICAL– Shortest time requirement on one end of strip: 30 sec– Longest time requirement on the other: 2 min

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• Principle of chemical reactions– False negative reactions– False positive reactions– Color interferences

• Alternative testing: used to confirm results that you may think are invalid due to– Interfering substance– Color interference (called color masking)

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Care and Storage (pg 4)

Confirmatory Testing (pg 6)

Reading assignment:

Textbook, chapter 7

Page 124-130

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Confirmatory Testing

• Alternative testing establishes the correctness or accuracy of another procedure

• Often used when urine is highly pigmented– Bilirubin reagent strip ictotest

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Confirmatory Testing

• Characteristics:– Differ in sensitivity

• Ictotest vs Bilirubin reagent strip

– Differ in specificity• SSA vs Protein reagent strip• Clinitest vs Glucose reagent strip

– Differ in methodology/reaction

Ideallywant all 3

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Differ in Specificity

• Clinitest reacts with all reducingsubstances

• Glucose reagent strip reacts with only one reducing substance: glucose

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10 reagent strip tests– Specific gravity– pH– Protein– Glucose– Ketones– Blood– Bilirubin– Urobilinogen– Nitrite– Leukocyte Esterase

• Purpose of the test• What is normal• What is abnormal• Reaction• Causes of invalid

results

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Specific Gravity: Purpose• Evaluates the concentrating and diluting

ability of the kidney– Density is related to the amount of

substances (solutes) in solution

– Increased density ~ increased solute in solution ~ hypertonic urine ~ concentrated urine

– Decreased density ~ decreased solute in solution ~ hypotonic urine ~ dilute urine

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Specific Gravity: Normal • Normal: 1.002 – 1.035

• Majority of urines: 1.010 – 1.025

• Physiologically impossible: 1.000>1.040

• Dependent upon hydration status

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Specific Gravity: Terms• Isosthenuria

– Fixed at 1.010– Renal tubules lost absorption and secreting capability

• Hypersthenuria– Increased specific gravity– Concentrated urine

• Hyposthenuria– Decreased specific gravity– Dilute urine

Sensitivity issues:Pregnancy testing

Urinary tract infection

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Specific Gravity: Methods

• Methods of measurement– Reagent strip test: indicates ionic solutes– Refractometer: indicates amount of total solutes

• Two functions of the kidney– Maintain water balance – Maintain electrolyte homeostasis

Performed by renal tubules through concentrating and diluting; reabsorbing and secreting water and electrolytes (ionic)

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Specific Gravity: Reaction • Based on a change in the pKa of a

polyelectrolyte on the reagent pad

• Increased ions in solution causes the polyelectrolyte on the pad to produce free H+

• Free H+ cause a change in pH on the reagent pad

• Change in pH: bromthymol blue indicator

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Specific Gravity: Reaction

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Specific Gravity

• Sensitivity: 1.000

• Specificity: detects only ionic substances– Radiographic dye– Mannitol– Glucose

Does not interfere

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pH: Purpose• Kidneys regulate body’s acid-base

balance by selective handling of H+ and HCO3-

– Urine pH reflects acid-base status of body

• Treatment protocol may require urine pH be maintained at a specific pH

(Aids in identification of crystals (microscope))

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pH: Normal

• Normal: ranges from 4.5 – 8.0

• First morning void: acidic

• Physiologically impossible: <4.5>8.0

1. Urine not handled properly

2. Old urine

3. Treatment induced

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pH: Interpretation

• Made in conjunction with – Acid-base status– Renal function – Presence of infection in urinary tract– Diet: high protein, low protein– Medications– Age of urine sample

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pH: Abnormal

• Acid– Respiratory acidosis– High protein diet– Starvation– UTI

• Alkaline– Respiratory alkalosis– Vegetarian diet– Renal tubular acidosis– UTI

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pH: Reaction

• Double indicator system– Methyl red– Bromthymol blue

• Amount of free H+ influences acidity of urine and cause pH indicator to change color

Needed to measure the wide pH range: acid to alkaline

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pH:

• Invalid test results due to:– Improper handling of urine sample

– Contamination of urine vessel prior to collection

– ‘Run-over’ phenomenon

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Protein: Purpose• Normal kidneys secrete LITTLE protein

<15 mg/dl (or <150 mg/24 hours)

• The protein that is found in urine comes from– Bloodstream– Urinary tract

• Proteinuria is an indicator of early renal disease

• Proteinuria also caused by non-renal disease

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Renal Cause of Proteinuria: • Glomerular damage:

– Most serious cause of proteinuria– Most common cause of proteinuria– Glomerulonephritis– Nephrotic Syndrome

• Tubular dysfunction:– Reabsorption capability decreased– Toxin exposure, inherited disorder– Fancon’s syndrome: heavy metal poisoning

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Classification of Proteinuria

• Functional• Orthostatic (postural)• Transient• Pathologic

– Pre-renal (overflow)– Renal: glomerular– Renal: tubular– Post-renal

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Protein: Methods

• Reagent strip test• SSA test• Foam test• Micro-albumin test

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Protein: Reagent Strip

• The reagent pad is held at a constant pH of 3 by a buffer

• Proteins (anions) in solution cause anindicator dye to release H+ causing a colorchange

• ‘Protein error of indicators’

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Protein: Reagent Strip• Sensitivity: ~ 10-25 mg/dl• Specificity: reacts with albumin

– False positive: highly alkaline urine (pH > 8.0)– False negative:

Dilute urine Presence of other proteins

(Tamm-Horsfall, globulins, myoglobin,

free light chains, hemoglobin)

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Protein: SSA (Exton’s Test)

• Sulfosalicylic Acid (SSA) Precipitation Test

• Acid will precipitate proteins out of solution causing the solution to become cloudy

• Amount of cloudiness is related to the amount of protein present

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• Amount of cloudiness is evaluated, thus must use centrifuged urine

• Sensitivity: 5-10 mg/dl• Specificity: detects all protein

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• False positive results: – Radiographic dyes– Turbid urine– Uncentrifuged urine

• False negative results:– Highly alkaline urine– Dilute urine

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Protein: Foam test

• Shake aliquot of urine and observe color of resulting foam

• White foam: protein present

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Protein: Micro-albumin test

• Measures very low concentration of albumin (better sensitivity than reagent strip test for albumin)

• Management of diabetic patient

• Methods vary: reagent strip test,immunochemical reaction

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Glucose: Purpose• Healthy normal urine does not contain

glucose

• Normally, glucose is filtered by the glomerulus and is reabsorbed back into the bloodstream through active transport mechanism

• Glucose in urine is pathologic

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Glucose: Purpose• Glucosuria

Glycosuria

• Caused by renal and non-renal disease– Pre-renal glycosuria: plasma glucose level

exceeds renal threshold (diabetes mellitus)

– Renal glycosuria: plasma glucose level below renal threshold, but tubules cannot reabsorb glucose back into bloodstream

Terms used interchangeably

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Reducing Substances: Purpose• Reducing Substances:

– Glucose– Other sugars: galactosemia (inherited

metabolic disorder)

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Glucose, Reducing Substances

• Normal: negative• Abnormal:

– Diabetes mellitus: glucose– Impaired renal tubular reabsorption: glucose

– Inborn error of metabolism: galactosemia

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Methods

• Reagent strip: detects only glucose

• Copper Reduction: detects reducing substances

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Glucose: Reagent Strip

• Detects only glucose• Double sequential enzyme reaction

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Glucose: Reagent Strip

• Sensitivity: ~ 30 mg/dl• Specificity:

– Reacts only with glucose– False positive:

• Strong oxidizing agents (bleach)• Peroxides

– False negative: • Ascorbic acid (reducing agent)• Improperly stored urine: glycolysis

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Clinitest Reaction

• Copper Reduction Test:– Reducing substances are able to reduce

copper sulfate to cuprous oxide

– Pass-through phenomenon

– All children <2 years: metabolic disorder(galactosemia)

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Clinitest Reaction

• Sensitivity: ~ 250 mg/dl• Specificity:

– Reacts with all reducing substances– Reducing sugars: glucose, galactose,

fructose, lactose, maltose (NOT SUCROSE)

– False positive: any reducing substance(Ascorbic acid)

– False negative: radiographic dye

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Ketones: Purpose

• Ketones are intermediary products of fat metabolism

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Ketones

• Three ketone bodies– Acetone 2%– Acetoacetic acid 20%– Beta-hydroxybutyric acid 78%

• Characteristic ‘fruity breath’ ~ acetone

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Ketones: Normal

• Normal: negative

• Abnormal:– Inability to utilize carbohydrates– Excessive loss of carbohydrates– Inadequate intake of carbohydrates

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Ketones: Methods

• Reagent strip

• Acetest: tablet test

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Ketones: Method

• Glycine: also measures acetone– Reagent strip: check package insert– Acetest tablets: contain glycine

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Ketones• Reagent strip

– Sensitivity: 5-10 mg/dl– Specificity: acetoacetic acid and/or acetone

• False positive: highly pigmented urine• False negative: improper specimen handling

• Acetest– Specificity: acetoacetic acid and acetone

• False positive: highly pigmented urien• False negative: improper specimen handling

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Blood: Purpose

• Blood in urine indicates pathology

• Two forms found in urine– Intact RBC– Hemolyzed RBC

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Blood: Terms

• Hematuria

• Hemoglobinuria

• Myoglobinuria

All will give a positive blood reaction

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Blood: Reagent strip

• Test can detect hemolyzed RBC

• Heme moiety imparts peroxidase activity and catalyzes the reaction

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Blood

• Sensitivity• Specificity

– Intact RBC– Hemolyzed RBC (hemoglobin)– Myoglobin

– False positives: myoglobin, oxidizing agents– False negatives: ascorbic acid

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Blood:

• Correlate reagent strip results– Microscopic findings– Color and clarity

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Bilirubin and Urobilinogen

• Bilirubin in urine is always pathologic: liver disease

• Urobilinogen in urine: normal to have a small amount: 0.2 – 1.0 mg/dl

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Three mechanisms

• Pre-hepatic: liver is healthy

• Hepatic: liver disease

• Post-hepatic: liver is healthy, obstruction indicated

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Bilirubin: Methods

• Reagent strip• Ictotest: tablet test• Foam test

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Bilirubin: Methods

• Reagent strip• Ictotest: tablet test

• Same reaction• Same specificity: conjugated bilirubin

– False positive: urine color– False negative: low concentration, ascorbic

acid, improper specimen handling

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Bilirubin: Methods

• Reagent strip• Ictotest: tablet test

• Sensitivity differsReagent strip: ~0.5 mg/dlIctotest: 0.05 – 0.1 mg/dl

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Bilirubin: Methods

• Possible to have a negative reagent strip test and positive ictotest– Difference in sensitivity levels

• Always perform Ictotest when– Urine bilirubin test specifically ordered– Urine appearance is amber: even if bilirubin

reagent strip test is negative– Positive reagent strip test

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Bilirubin: Foam Test

• Shake urine and observe resulting foam• Yellow foam = bilirubin

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Urobilinogen: Methods

• Reagent strip test– Two reactions dependent upon manufacturer

• Para-dimethylaminobenzaldehyde• Diazonium salt

– Cannot determine absence of UBG

• Watson-Schwartz assay

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Urobilinogen: Methods• Para-dimethylaminobenzaldehyde

– Sensitivity: 0.2 mg/dl– Specificity:

• False positive: any ‘Ehrlich reactive compound’; color masking; urine at body temp

• False negative: improper specimen handling

• Diazonium salt– Sensitivity: 0.4 mg/dl– Specificity: reacts only with UBG

• False positive: color masking• False negative: improper specimen handling

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Urobilinogen: Watson Schwartz

• Classic method used to differentiateurobilinogen from porphobilinogen using adifferential extraction method

• Para-dimethylaminobenzaldehyde

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Nitrite: Purpose

• Bacteria that contain a specific enzyme can reduce dietary nitrates to nitrites

• Rapid screening test for UTI

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Nitrite: Normal


• Abnormal:– Cystitis: bladder– Pyelonephritis: kidney

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Nitrite: Method• Reagent strip test

Nitrite + aromatic amine diazonium saltDiazonium salt + aromatic compound pink color

• Sensitivity: 0.06-0.1 mg/dl nitrite~ 10,000 organisms

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Nitrite: Method• Reagent strip test

Specificity:– False positive: improper specimen handling; color

masking– False negative: bacteria cannot reduce nitrates

Bladder time not sufficient: need 4 hoursLow nitrite levelsAscorbic acidAntibiotic inhibition of bacteriaFurther reduction of nitrites to nitrogen

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Leukocyte Esterase: Purpose

• Increased WBC in urine is pathologic– Indicates inflammation, infection

• Neutrophils most common type of WBC found in urine

• Can detect intact WBC and lysed WBC

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Leukocyte Esterase: Normal


• Abnormal:– Bacterial infection:

cystitis, pyelonephritis, urethritis– Non-bacterial infection: yeast, trichomonas

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Leukocyte Esterase: Method

• Reagent strip: Granules in cytoplasm of WBC contain an enzyme (esterase)

Ester –esterase aromatic compoundAromatic compound + diazonium salt Purple colored complex

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Leukocyte Esterase

• Sensitivity: 5-15 WBC/hpf

• Specificity:– False positive: vaginal contamination; color

masking– False negative: strong oxidizing agents

(bleach); lymphocytes (no granules)

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Chemical Examination of Urine