1. Patient Project: Urine Test Strips
Week 9
Today’s Agenda:
Review patient project.
Review urine test strips.
Develop the day’s problem.
Analyze test strips.
Analyze patient samples

2. The Patient Project 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

3. Terminology of Analysis
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 “semi-quantitative” mean?

4. 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.

5. 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

6. Urine Test Strip Pre-lab
questions
class results

7. Medical Waste A New Waste Stream
And Gloves
are a
MUST WEAR
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).

8. 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.

9. Let′s think!
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.

10. Test three known urine samples and then your own patient’s sample. .
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.
normal and abnormal samples.
Your group will need data from all five tests. Decide in your group who is going to do what.
You have 20 minutes

11. Urine pH Reagent Strip Methodology
Bromthymol Blue
Methyl Red H+
pH 4.4 = Red
pH 6.2 = Yellow
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
8.0
7.5
8.5

12. Urine pH Reagent Strip Methodology
5.0
6.0
6.5
7.0
8.0
7.5
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.

13. Urine Glucose Reagent Strip Methodology
Glucose Oxidase
Glucose
Peroxidase
Gluconic acid
+ H2O2
H2O K+I-
K+ + H2O + I2
H2O K+IO-
2H+
K+IO-
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:

14. Urine Glucose Reagent Strip Methodology
Negative
100
250
500
2000
1000
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.

15. Urine Ketone Reagent Strip Methodology
Acetoacetate
In urine sample
sodium nitroprusside
Iron-acetoacetate complex
+ Na+CN- + NO(g)
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.

16. Urine Ketone Reagent Strip Methodology
Acetoacetate
In urine sample
sodium nitroprusside
on strip pad
Iron-acetoacetate complex
+ Na+CN- + NO(g)
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
Small 40
Moderate
160
Large 80
mg/dL

17. 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
N-Acetyl-
cysteine
Methyldopa

18. Urine Protein Reagent Strip Methodology
H2N-Protein in urine
Protonated Tetrabrom-phenol Blue
(Yellow)
Anionic Tetra-bromphenol Blue
(Bluish-green)
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.

19. Urine Protein Reagent Strip Methodology
H2N-Protein in urine
Protonated Tetrabrom-phenol Blue
(Yellow)
Anionic Tetra-bromphenol Blue
(Bluish-green)
Semi-quantitative interpretation is by color after 60 seconds:
Negative
Trace 30
100
>2000
300
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.

20. Urine Blood Reagent Strip MethodologyHb
3,3’,5,5’- Tetramethylbenzidine Reduced
(Yellow)
Peroxidase
activity
Cumene
hydroperoxide
2-Phenyl
-2-propanol
3,3’,5,5’- Tetramethylbenzidine 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:
Non-
Hemolyzed
Trace
Negative
Small
Large
Moderate

21. Urine Blood Reagent Strip Methodology
Non-
Hemolyzed
Trace
Negative
Small
Large
Moderate
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.

22. pH measurement what do you know about pH?
A highly acidic urine pH occurs in:
Acidosis
Uncontrolled diabetes
Diarrhea
Starvation and dehydration
Respiratory diseases in which carbon dioxide retention occurs and
acidosis develops
A highly alkaline urine occurs in:
Urinary tract obstruction
Pyloric obstruction
Salicylate intoxication
Renal tubular acidosis
Chronic renal failure
Respiratory diseases that involve hyperventilation (blowing off carbon dioxide and the development of alkalosis)
what do you know about pH?
why is pH measurement valuable?

23. Using a pH electrode with Logger Pro.
Let′s think!
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.

24. 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.

25. Let′s think!
Glucose Solutions
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 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.

26. In your groups discuss how you expect to answer these questions.
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
2000
1000
mg/dL

27. 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.

28. 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.
How precise is the color method for reading pH?
How precise is the color method for measuring [glucose]?
Does the glucose strip give positive readings with other sugars?
How does household bleach affect the test strips? What problems might be encountered if the strips are mishandled?

29. Your Final Challenge Perform the reactions
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.
normal and abnormal samples.
test reagents.
pH meter and electrode.
Implement your designed experimental procedures!
You have 90 minutes