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

3

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

5

Protein: Methods

• Reagent strip test

• SSA test

• Foam test

• Micro-albumin test

6

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’

7

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)

8

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

9

Protein: SSA (Exton’s Test)

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

• Sensitivity: 5-10 mg/dl

• Specificity: detects all protein

10

Protein: SSA (Exton’s Test)

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

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

11

Protein: Foam test

• Shake aliquot of urine and observe color of resulting foam

• White foam: protein present

12

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

13

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

14

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

15

Reducing Substances: Purpose

• Reducing Substances:– Glucose– Other sugars: galactosemia (inherited

metabolic disorder)

16

Glucose, Reducing Substances

• Normal: negative

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

– Inborn error of metabolism: galactosemia

17

Methods

• Reagent strip: detects only glucose

• Copper Reduction: detects reducing substances

18

Glucose: Reagent Strip

• Detects only glucose

• Double sequential enzyme reaction

19

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

20

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)

21

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