Chemical
Examination
of Urine
Ricki Otten MT(ASCP)SC
uotten@unmc.edu
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
2
Historical Perspective: Urinalysis
• Physical examination of urine
– Odor
– Taste
– Color
– Clarity
3
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
4
Historical Perspective
• Microscopic examination of urine
– Not until invention of the microscope
– Then clinical usefulness realized
5
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
6
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
7
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 interpretation8
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
9
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)
10
Care and Storage (pg 4)
Reading assignment:
Textbook, chapter 7
Page 124-130
Confirmatory Testing (pg 6)
11
Confirmatory Testing
• Alternative testing establishes the
correctness or accuracy of another
procedure
• Often used when urine is highly pigmented
– Bilirubin reagent strip  ictotest
12
Confirmatory Testing
• Characteristics:
– Differ in sensitivity
• Ictotest vs Bilirubin reagent strip
– Differ in specificity
• SSA vs Protein reagent strip
• Clinitest vs Glucose reagent strip
Ideally
want
all 3
– Differ in methodology/reaction
13
Differ in Specificity
• Clinitest reacts with all reducing
substances
• Glucose reagent strip reacts with only
one reducing substance: glucose
14
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
15
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
16
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
17
Specific Gravity: Terms
• Isosthenuria
– Fixed at 1.010
– Renal tubules lost absorption and secreting capability
• Hypersthenuria
– Increased specific gravity
– Concentrated urine
• Hyposthenuria
Sensitivity issues:
Pregnancy testing
Urinary tract infection
– Decreased specific gravity
– Dilute urine
18
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)
19
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
20
Specific Gravity: Reaction
21
Specific Gravity
• Sensitivity: 1.000
• Specificity: detects only ionic substances
– Radiographic dye
– Mannitol
– Glucose
Does not interfere
22
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))
23
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
24
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
25
pH: Abnormal
• Acid
–
–
–
–
Respiratory acidosis
High protein diet
Starvation
UTI
• Alkaline
–
–
–
–
Respiratory alkalosis
Vegetarian diet
Renal tubular acidosis
UTI
26
pH: Reaction
• Double indicator system
– Methyl red
– Bromthymol blue
Needed to measure
the wide pH range:
acid to alkaline
• Amount of free H+ influences acidity of
urine and cause pH indicator to change
color
27
pH:
• Invalid test results due to:
– Improper handling of urine sample
– Contamination of urine vessel prior to
collection
– ‘Run-over’ phenomenon
28
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
29
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
30
Classification of Proteinuria
•
•
•
•
Functional
Orthostatic (postural)
Transient
Pathologic
– Pre-renal (overflow)
– Renal: glomerular
– Renal: tubular
– Post-renal
31
Protein: Methods
•
•
•
•
Reagent strip test
SSA test
Foam test
Micro-albumin test
32
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’
33
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)
34
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
35
Protein: SSA (Exton’s Test)
• Amount of cloudiness is evaluated, thus
must use centrifuged urine
• Sensitivity: 5-10 mg/dl
• Specificity: detects all protein
36
Protein: SSA (Exton’s Test)
• False positive results:
– Radiographic dyes
– Turbid urine
– Uncentrifuged urine
• False negative results:
– Highly alkaline urine
– Dilute urine
37
Protein: Foam test
• Shake aliquot of urine and observe color
of resulting foam
• White foam: protein present
38
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
39
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
40
Glucose: Purpose
• Glucosuria
Glycosuria
Terms used
interchangeably
• 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
41
Reducing Substances: Purpose
• Reducing Substances:
– Glucose
– Other sugars: galactosemia (inherited
metabolic disorder)
42
Glucose, Reducing Substances
• Normal: negative
• Abnormal:
– Diabetes mellitus: glucose
– Impaired renal tubular reabsorption: glucose
– Inborn error of metabolism: galactosemia
43
Methods
• Reagent strip: detects only glucose
• Copper Reduction: detects reducing
substances
44
Glucose: Reagent Strip
• Detects only glucose
• Double sequential enzyme reaction
45
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
46
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)
47
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
48
Ketones: Purpose
• Ketones are
intermediary products
of fat metabolism
49
Ketones
• Three ketone bodies
– Acetone
– Acetoacetic acid
– Beta-hydroxybutyric acid
2%
20%
78%
• Characteristic ‘fruity breath’ ~ acetone
50
Ketones: Normal
• Normal: negative
• Abnormal:
– Inability to utilize carbohydrates
– Excessive loss of carbohydrates
– Inadequate intake of carbohydrates
51
Ketones: Methods
• Reagent strip
• Acetest: tablet test
52
Ketones: Method
• Glycine: also measures acetone
– Reagent strip: check package insert
– Acetest tablets: contain glycine
53
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
54
Blood: Purpose
• Blood in urine indicates pathology
• Two forms found in urine
– Intact RBC
– Hemolyzed RBC
55
Blood: Terms
• Hematuria
• Hemoglobinuria
All will give a
positive blood
reaction
• Myoglobinuria
56
Blood: Reagent strip
• Test can detect hemolyzed RBC
• Heme moiety imparts peroxidase activity
and catalyzes the reaction
57
Blood
• Sensitivity
• Specificity
– Intact RBC
– Hemolyzed RBC (hemoglobin)
– Myoglobin
– False positives: myoglobin, oxidizing agents
– False negatives: ascorbic acid
58
Blood:
• Correlate reagent strip results
– Microscopic findings
– Color and clarity
59
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
60
Three mechanisms
• Pre-hepatic: liver is healthy
• Hepatic: liver disease
• Post-hepatic: liver is healthy, obstruction
indicated
61
Bilirubin: Methods
• Reagent strip
• Ictotest: tablet test
• Foam test
62
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
63
Bilirubin: Methods
• Reagent strip
• Ictotest: tablet test
• Sensitivity differs
Reagent strip: ~0.5 mg/dl
Ictotest:
0.05 – 0.1 mg/dl
64
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
65
Bilirubin: Foam Test
• Shake urine and observe resulting foam
• Yellow foam = bilirubin
66
Urobilinogen: Methods
• Reagent strip test
– Two reactions dependent upon manufacturer
• Para-dimethylaminobenzaldehyde
• Diazonium salt
– Cannot determine absence of UBG
• Watson-Schwartz assay
67
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
68
Urobilinogen: Watson Schwartz
• Classic method used to differentiate
urobilinogen from porphobilinogen using a
differential extraction method
• Para-dimethylaminobenzaldehyde
69
Nitrite: Purpose
• Bacteria that contain a specific enzyme
can reduce dietary nitrates to nitrites
• Rapid screening test for UTI
70
Nitrite: Normal
• Normal: negative
• Abnormal:
– Cystitis: bladder
– Pyelonephritis: kidney
71
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
72
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
73
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
74
Leukocyte Esterase: Normal
• Normal: negative
• Abnormal:
– Bacterial infection:
cystitis, pyelonephritis, urethritis
– Non-bacterial infection: yeast, trichomonas
75
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
76
Leukocyte Esterase
• Sensitivity: 5-15 WBC/hpf
• Specificity:
– False positive: vaginal contamination; color
masking
– False negative: strong oxidizing agents
(bleach); lymphocytes (no granules)
77
78