Chemical Examination of Urine

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Chemical Examination of Urine

• Ricki Otten MT(ASCP)SC
• uotten_at_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 status
• of 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)
Reading assignment Textbook, chapter 7 Page
124-130
Confirmatory Testing (pg 6)
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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
• gt1.040
• 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 bodys 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 lt4.5
• gt8.0

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
• lt15 mg/dl (or lt150 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
• Fancons 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 an
• indicator dye to release H causing a color
• change
• 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 gt 8.0)
• False negative
• Dilute urine
• Presence of other proteins
• (Tamm-Horsfall, globulins, myoglobin,
• free light chains, hemoglobin)

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Protein SSA (Extons 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 (Extons 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 (Extons 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 lt2 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 differs
• Reagent strip 0.5 mg/dl
• Ictotest 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 differentiate
• urobilinogen from porphobilinogen using a
• differential 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

• Normal negative
• Abnormal
• Cystitis bladder
• Pyelonephritis kidney

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Nitrite Method

• Reagent strip test
• Nitrite aromatic amine ? diazonium salt
• Diazonium 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 hours
• Low nitrite levels
• Ascorbic acid
• Antibiotic inhibition of bacteria
• Further 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

• Normal negative
• 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 compound
• Aromatic 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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