Chemical Examination of UrinePart I:

Introduction, Specific Gravityand pH

Ricki Otten MT(ASCP)SC

uotten@unmc.edu

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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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Reagent Strip Testing

• 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 that

takes place upon contact with urine

• Chemical reaction causes the color of the pad to

change

• Color compared to a color chart for interpretation

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Reagent Strip Testing

• 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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Reagent Strip Testing

• 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 reducing

substances

• 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

>8.01. 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