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Chemical Examination of Urine
Part II:Protein, Glucose
Ricki Otten MT(ASCP)SC
uotten@unmc.edu
2
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
4
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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Protein: SSA (Exton’s Test)
• Amount of cloudiness is evaluated, thus must use centrifuged urine
• Sensitivity: 5-10 mg/dl
• Specificity: detects all protein
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Protein: SSA (Exton’s Test)
• 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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