Objectives:
Chemical Examination of
Urine
Part I:
Introduction, Specific Gravity
and pH
• Review the objectives on page 1 and 2
of the lecture handout
• Objectives marked with ‘*’ will not be
tested over during student lab rotation
Ricki Otten MT(ASCP)SC
uotten@unmc.edu
2
Historical Perspective: Urinalysis
Historical Perspective
• Physical examination of urine
• Chemical examination of urine
– Odor
– Taste
– Color
– Clarity
– 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
3
4
Historical Perspective
Reagent Strip Testing
• Microscopic examination of urine
•
•
•
•
•
•
– Not until invention of the microscope
– Then clinical usefulness realized
Technology and necessity
Chemical reactions ‘miniaturized’
Required less urine
Test results within minutes
Easy to perform
Increased test utilization
5
CLS 426 Urine and Body Fluid Analysis: Student Lab Rotation
Chemical Exam of Urine Lecture
Brunzel, 2nd Ed, page 124 6
1
Reagent Strip Testing
Reagent Strip Testing
• Ideal qualitative screening tool
• Chemically impregnated absorbent
pads attached to an inert plastic strip
– Sensitive: Low concentration of substances
Negative result = normal
• Each pad is a specific chemical reaction that
takes place upon contact with urine
– 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
• Chemical reaction causes the color of the pad to
change
• Color compared to a color chart for interpretation8
Reagent Strip Testing
Reagent Strip Testing
• Qualitative or semi-quantitative results
• Principle of chemical reactions
– Concentration units (mg/dl)
– Negative, small, moderate large
– Negative, 1+, 2+, 3+, 4+
– False negative reactions
– False positive reactions
– Color interferences
• Timing of chemical reactions is CRITICAL
• Alternative testing: used to confirm results that
you may think are invalid due to
– Shortest time requirement on one end of strip: 30 sec
– Longest time requirement on the other: 2 min
– Interfering substance
– Color interference (called color masking)
9
Care and Storage (pg 4)
10
Confirmatory Testing
• Defined: Alternative testing establishes the
correctness or accuracy of another
procedure
Reading assignment:
Textbook, chapter 7
Page 124-130
• Often used when urine is highly pigmented
or when results are suspected to be
incorrect
Confirmatory Testing (pg 6)
– Bilirubin reagent strip Æ ictotest
11
CLS 426 Urine and Body Fluid Analysis: Student Lab Rotation
Chemical Exam of Urine Lecture
12
2
Confirmatory Testing
Differ in Specificity
• Characteristics:
• Clinitest reacts with all reducing
substances
– 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
• Glucose reagent strip reacts with only
one reducing substance: glucose
– Differ in methodology/reaction
13
Specific Gravity: Purpose
10 reagent strip tests
–
–
–
–
–
–
–
–
–
–
Specific gravity
pH
Protein
Glucose
Ketones
Blood
Bilirubin
Urobilinogen
Nitrite
Leukocyte Esterase
14
•
•
•
•
•
• Evaluates the concentrating and diluting
ability of the kidney
Purpose of the test
What is normal
What is abnormal
Reaction
Causes of invalid
results
– 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
15
16
Specific Gravity: Terms
Specific Gravity: Normal
• Isosthenuria
• Normal: 1.002 – 1.035
– Fixed at 1.010
– Renal tubules lost absorption and secreting capability
• Majority of urines: 1.010 – 1.025
• Hypersthenuria
– Increased specific gravity
– Concentrated urine
• Physiologically impossible: 1.000
>1.040
• Hyposthenuria
• Dependent upon hydration status
Sensitivity issues:
Pregnancy testing
Urinary tract infection
– Decreased specific gravity
– Dilute urine
17
CLS 426 Urine and Body Fluid Analysis: Student Lab Rotation
Chemical Exam of Urine Lecture
18
3
Specific Gravity: Reaction
Specific Gravity: Methods
• Based on a change in the pKa of a
polyelectrolyte on the reagent pad
• 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)
• 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
19
Specific Gravity: Reaction
20
Specific Gravity
• Sensitivity: 1.000
• Specificity: detects only ionic substances
– Radiographic dye
– Mannitol
– Glucose
Does not interfere
21
22
pH: Purpose
pH: Normal
• Kidneys regulate body’s acid-base
balance by selective handling of H+ and HCO3-
• Normal: ranges from 4.5 – 8.0
– Urine pH reflects acid-base status of body
• First morning void: acidic
• Treatment protocol may require urine pH be
maintained at a specific pH
• Physiologically impossible: <4.5
>8.0
(Aids in identification of crystals (microscope))
1. Urine not handled properly
2. Old urine
3. Treatment induced
23
CLS 426 Urine and Body Fluid Analysis: Student Lab Rotation
Chemical Exam of Urine Lecture
24
4
pH: Interpretation
pH: Abnormal
• Made in conjunction with
• Acid
–
–
–
–
– Acid-base status
– Renal function
– Presence of infection in urinary tract
– Diet: high protein, low protein
– Medications
– Age of urine sample
• Alkaline
Respiratory acidosis
High protein diet
Starvation
UTI
–
–
–
–
Respiratory alkalosis
Vegetarian diet
Renal tubular acidosis
UTI
25
pH: Reaction
pH:
• Double indicator system
– Methyl red
– Bromthymol blue
26
• Invalid test results due to:
– Improper handling of urine sample
Needed to measure
the wide pH range:
acid to alkaline
– Contamination of urine vessel prior to
collection
H+
• Amount of free
influences acidity of
urine and cause pH indicator to change
color
– ‘Run-over’ phenomenon
27
CLS 426 Urine and Body Fluid Analysis: Student Lab Rotation
Chemical Exam of Urine Lecture
28
5