Transcript GenChem

GenChem
Week 9
Patient Project:
Urine Test Strips
Today’s Agenda:
 Review patient project.
 Review urine test strips.
 Develop the day’s problem.
Analyze test strips.
Analyze patient samples
The Patient Project
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 Patient case studies
 Patient interview data
 Urine analysis (test strips)
 CK and LD tests
Glucose tests
Ion-selective electrodes
 Diagnosis and presentation as a poster
Let′s think!
Terminology of Analysis
In your groups
discuss what the following
terms mean to you
qualitative analysis
quantitative analysis
How do they apply to medical
diagnosis?
What does the term “semiquantitative” mean?
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Let′s think!
Test Strips
In your groups review your
responses to the following
pre-lab question and come
up with a consensus answer.
Describe in your own words how a
test strip works and what kind of
information it provides.
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Reagent Strip Urinalysis
Devices designed to perform chemical tests quickly, easily and
reliably and to generate easy-to-interpret results.
Dozens of tests are available in reagent strip format
We will examine five common tests. For each we will
address:
What each test is designed to detect or measure
The chemistry behind each test
How individual test results are interpreted
Examples of limitations and interferences
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Urine Test
Strip Pre-lab
questions
class results
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Medical Waste And Gloves
A New Waste Stream are a
This experiment introduces medical
waste.
The urine samples, and everything
the contact, are considered medical
waste.
Liquid medical waste goes in the
liquid waste bucket in the hood.
Solid medical waste goes in the
special biohazard waste container
(with the iconic red bag).
MUST WEAR
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Biohazard Cleanup
At the conclusion of all experimental work all work
surfaces must be decontaminated using the Amphyl
antimicrobial agent.
A commercial product that disinfects.
Wear gloves!
The Amphyl is located in a squeeze bottle at the
end of the lab bench. There will also be a dedicated
sponge to use.
Squirt liberally and wipe. Squirt the Amphyl on the
work area and wipe with the sponge.
When done, leave the bottle and sponge at the end
of the workbench.
Let′s think!
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Patient Analysis
Use the strips to test three known
urine samples and then your own
patient’s sample.
The samples are expensive. To minimize waste,
A limited amount of each sample is put out in the lab. Your instructor
will show you where they are located.
Take only what you need.
Use a clean transfer pipet for each sample.
Take some of the sample up in the pipet, spot it to the pad and then
dispose of the pipet. Do not put any sample that may remain in the
pipet back into the sample bottle.
All used transfer pipets are to be disposed of as medical waste.
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Let′s explore!
Your First Challenge
Test three known urine samples and then
your own patient’s sample. .
Available resources:
up to 20 test strips.
patient sample.
Your group will need
data from all five tests.
Decide in your group
who is going to do what.
normal and abnormal samples.
You have
20 minutes
GenChem
Urine pH Reagent Strip Methodology
Methyl Red
H+
H+
Bromthymol
Blue
pH 6.2 = Yellow
pH 4.4 = Red
H+
H+
pH 6.0 = Yellow
pH 7.6 = Blue
Semi-quantitative interpretation by color after 60 seconds in increments of 0.5
pH units. Note: pH range limited to 5-9, which is the range of physiological
/clinical significance.
5.0
6.0
6.5
7.0
7.5
8.0
8.5
GenChem
Urine pH Reagent Strip Methodology
5.0
6.0
6.5
7.0
7.5
8.0
8.5
Interference and Limitations:
Bacterial growth in a specimen may cause an alkaline shift
from conversion of urea (neutral) to ammonia (basic).
Excessively wet strips may permit the acidic or basic
buffers of other pads to run into the pH pad, resulting in a
“false” interpretation.
Accurate interpretation of deeply colored (dark yellow)
urine samples maybe difficult.
GenChem
Urine Glucose Reagent Strip Methodology
Glucose Oxidase
+ H2O2
Gluconic acid
Glucose
Peroxidase
H2O2 +
K + I-
H2O + K+IO-
Peroxidase
K+IO-
2H+
K+ + H2O + I2
The reagent pad contains the enzymes glucose oxidase and peroxidase along with I-. In
the presence of urine glucose, glucose oxidase forms hydrogen peroxide (H2O2 ) and
gluconic acid. Peroxidase then catalyzes the oxidation of I- with the H2O2 formed in the
first reaction.
The enzyme glucose oxidase is very specific for glucose, hence the exquisite glucose
selectivity of the test.
Semi-quantitative interpretation is by color after 30 seconds:
GenChem
Urine Glucose Reagent Strip Methodology
Negative
100
250
500
1000
2000
mg/dL
Interference and Limitations:
False positive results can occur in the presence of peroxides or other
oxidizing reagents (eg. cleaning agents) that oxidize the chromogen
independently of H2O2 production by the first enzymatic reaction
(uncoupling of the indicator reaction from the test reaction)
False negative results can occur with high concentrations of ascorbic
acid or ketones which inhibit the peroxidase reaction.
Urine samples must be at room temperature; low temperatures can
lead to artificially low readings.
Enzymes are sensitive to humidity and air.
Accurate interpretation of deeply colored (dark yellow) urine samples
maybe difficult.
GenChem
Urine Ketone Reagent Strip Methodology
sodium
nitroprusside
2-
Iron-acetoacetate complex
2Na+
+ Na+CN- + NO(g)
Acetoacetate
In urine sample
Alkaline Buffer
The reagent pad contains sodium nitroprusside with a strongly basic buffer.
Acetoacetate is a 1,3-dicarbonyl. In basic conditions, 1,3-dicarbonyls form
strong coordination complexes with iron that are intensely colored.
There are virtually no other 1,3-dicarbonyl compounds found in urine, so the
test is reasonably specific for acetoacetate.
GenChem
Urine Ketone Reagent Strip Methodology
sodium nitroprusside
on strip pad
2-
Iron-acetoacetate complex
2Na+
+ Na+CN- + NO(g)
Acetoacetate
In urine sample
Alkaline Buffer
Formation of the reddish-purple colored iron-acetoacetate coordination complex is
proportional to the amount of acetoacetate in the urine sample.
Semi-quantitative interpretation is by color after 40 seconds:
Negative
5
Trace
15
40
Small Moderate
80
Large
160
Large
mg/dL
GenChem
Urine Ketone Reagent Strip Methodology
Interference and Limitations:
False positive results may occur with some preservatives,
dehydration, L-Dopa metabolites, anti-hypertensive drugs
(such as methyldopa and captopril), and some sulfhydryl
containing drugs (eg. N-Acetylcysteine)
Captopril
Methyldopa
N-Acetylcysteine
GenChem
Urine Protein Reagent Strip Methodology
Protonated
Tetrabromphenol Blue
(Yellow)
H2N-Protein in urine
Anionic Tetrabromphenol
Blue
(Bluishgreen)
Detects protein in urine by pH indicator (Tetrabromphenol blue) color
shift.
Tetrabromphenol blue is buffered to pH 3 to give the protonated form
which is yellow.
If protein is present, the yellow protonated tetra-bromphenol blue is
shifted to the anionic form which is deep bluish-green in color.
GenChem
Urine Protein Reagent Strip Methodology
Protonated
Tetrabromphenol Blue
(Yellow)
H2N-Protein in urine
Anionic Tetrabromphenol
Blue
(Bluish-green)
Semi-quantitative interpretation is by color after 60 seconds:
Negative
Trace
30
100
300
>2000
mg/dL
Interference and Limitations:
False positive results maybe produced by quaternary ammonium
compounds used for cleaning and amido-amines in fabric softeners and
with highly alkaline urine which is seen in patients on alkaline
medications or with bacterial contamination of the urine sample.
False negative results can occur with high salt concentrations.
GenChem
Urine Blood Reagent Strip Methodology
Hb
Peroxidase
activity
Cumene
hydroperoxide
3,3’,5,5’Tetramethylbenzid
ine Reduced
(Yellow)
2-Phenyl
-2-propanol
3,3’,5,5’Tetramethylbenzid
ine Oxidized
(Dark-green)
Detects the presence of Hemoglobin (Hb) in urine.
Hemoglobin (Hb) functions as a peroxidase that catalyzes reduction
of peroxides in the presence of a hydrogen donor.
With intact red blood cells (RBCs), spotting occurs, since the RBCs
for aggregates (clumps) and the Hb remains inside the RBCs.
Qualitative interpretation is by color and spotting after 60 seconds:
Negative
NonHemolyzed
Trace
Hemolyzed
Trace
Small
Moderate
Large
GenChem
Urine Blood Reagent Strip Methodology
Negative
NonHemolyzed
Hemolyzed
Trace
Trace
Small
Moderate
Large
Interference and Limitations:
False positive can occur with oxidizing agents (peroxides,
bleach, and Betadine used in penile or perineal cleaning) and
microbial peroxidase.
False negative can occur with reducing agents (e.g. ascorbic
acid), formaldehyde, or very high urine specific gravity.
GenChem
pH measurement
A highly acidic urine pH occurs in:
• Acidosis
• Uncontrolled diabetes
• Diarrhea
• Starvation and dehydration
 what
do you
• Respiratory
diseases
in which carbon dioxide retention occurs and
• acidosis
develops
know
about pH?
A highly alkaline urine occurs in:
 why is pH
• Urinary tract obstruction
• Pyloric
obstruction
measurement
• Salicylate intoxication
valuable?
• Renal
tubular acidosis
• Chronic renal failure
• Respiratory diseases that involve hyperventilation (blowing off carbon
dioxide and the development of alkalosis)
Let′s think!
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Using a pH electrode with Logger Pro.
Make sure you know
How to calibrate the
electrode.
proper electrode care.
how to read the meter.
how to leave the setup
when done.
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Let′s think!
pH analysis
The question is how precise is the color
method for reading pH? In your group,
discuss how to answer this.
Recommended procedure for making solutions
to test.
put 100 mL of water in a 250 mL beaker.
Add pH buffers. A total of 10 drops should
be sufficient.
Mix the solution.
Spot the solution on the test strip and “read”
the pH.
Measure the actual pH.
Add more buffer and repeat.
Let′s think!
GenChem
Glucose Solutions
In your groups review your
responses to the following prelab question and come up with
a consensus answer.
Describe how you expect to go about using the stock
solution of glucose (2000 mg/dL) to evaluate the
glucose test strips. Create an outline of all the
procedures you expect to perform including the
amounts of materials you expect to be using.
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Let′s think!
Evaluating Interferences
In your groups discuss how
you expect to answer these
questions.
 Does the glucose strip give
positive readings with other
sugars? How specific for
glucose is it?
Negative
100
250
500
1000
2000
mg/dL
Let′s think!
Effect of Bleach
In your groups review your
responses to the following pre-lab
question and come up with a
consensus answer.
Describe how you expect to go about
evaluating the effect of bleach on the test
strips. Include a description of the
operations you expect to perform and the
amounts of materials you expect to use.
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Divvying up tasks
Your group will need data from all five tests.
Decide in your group who is going to do what.
Test Analysis. Answer a series of questions.
1. How precise is the color method for reading pH?
2. How precise is the color method for measuring [glucose]?
3. Does the glucose strip give positive readings with other sugars?
4. How does household bleach affect the test strips? What problems
might be encountered if the strips are mishandled?
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Let′s explore!
Your Final Challenge
Perform the reactions
Analyze the test strips and the samples.
Available resources:
up to 20 test strips.
patient sample.
Implement your
designed experimental
procedures!
normal and abnormal samples.
test reagents.
pH meter and electrode.
You have
90 minutes