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

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

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?

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.

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

Urine Test Strip Pre-lab questions class results

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).

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! 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.

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

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

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.

Urine Glucose Reagent Strip Methodology Glucose Oxidase Glucose Peroxidase Gluconic acid + H2O2 H2O2 + 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:

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.

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.

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

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

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.

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.

Urine Blood Reagent Strip Methodology Hb 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

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.

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?

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.

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! 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.

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

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.

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?

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