108.4

What are reagent strips?

Strips that consist of chemical-impregnated absorbent pads

What are some advantages in using in the Routine Urinalysis test?

Provide simple, rapid means for performing routine chemical tests on urine

Explain how chemical reactions are interpreted on reagent strips

Compare the color produced on the pad within time frame with a chart supplied by manufacturer

What is the difference between a semi-quantitative result and quantitative result?

Semi-quantitative uses degrees of readings such as 1+, 2+, 3+, and 4+
Quan uses actual numbers as the measured amount

List the 10 chemical tests on a urine dipstick

pH
Protein
Glucose
Ketones
Blood
Bilirubin
Urobilinogen
Nitrate
Leukocyte Esterase
Specific Gravity

Describe the proper technique for performing reagent strip testing.

Dip strip briefly into well-mixed specimen at room temperature
Remove excess urine by touching edge of strip to container as strip is withdrawn
Blot edge of strip on absorbent pad
Wait specified amount of time
Read using a good light source

List 4 steps in good dipping technique

Urine must be well mixed before testing with dipstick
Each pad must come in contact with the urine for the chemical reaction
Remove dipstick from urine immediately once the pad is saturated
Blot the strip on edge to remove excess urine to avoid run-off

List 6 improper technique errors.

Improper mixing of specimen
Keeping strip in the urine for too long
Not blotting the strip after dipping
Not keeping track of the time
Bad light source for reading test
Not properly comparing pads to color chart

Discuss how reagent strips should be handled and stored

Store with desiccant in an opaque, tightly sealed container
Remove strips immediately prior to use
Do not expose to volatile fumes
Store below 30°C
Do not use past the expiration date
Visually inspect for discoloration/deterioration

List four quality-control procedures routinely performed with reagent strip testing.

Run positive and negative controls, usually at the beginning of a shift
When a new lot of strips is opened
When results are questionable
When there are concerns over strip integrity

What is confirmatory testing and when should it be performed?

Confirmatory tests use different reagents or methodologies to detect the same substances as reagent strips with the same or greater sensitivity or specificity
May be available when questionable results are obtained

List the reasons for measuring urinary pH, and discuss their clinical applications

Acid-base content of the blood
Patient's renal function
Presence of a urinary tract infection
Renal calculi formation
High-protein diets=acidic urine
Low-protein diets=alkaline urine
Old specimen pH >8.5 warrants recollection

Discuss the principle of pH testing by reagent strip

Double indicator system Methyl Red - 4 to 6 pH
Bromthymol blue - 6 to 9 pH
Double due to wide range
Methyl Red + H+ = acid pH
Bromthymol blue - H+ = alkaline pH

List any interference or Source of Error in pH testing

No known substance interferes with testing
Runover from adjacent pads may alter pH

Differentiate between prerenal, renal, and postrenal proteinuria, and give clinical examples of each

Prerenal- conditions affecting the plasma, not the kidney - acute phase reactants, Multiple Myeloma
Renal - Glomerular or Tubular damage
Microalbuminuria present, Lupus, Glomerulonephritis
Post-Renal - Protein added in lower urinary - UTI, Prostate issues

Briefly explain the principle of the protein error of indicators. How does a highly alkaline urine affect this?

When the pH remains constant, certain indicators change color in the presence of protein; highly alkaline urine will override the reagent strip pH buffer, thus producing a color change related to pH, not protein.

State a pathological condition that would cause a negative reagent strip protein reaction and a positive sulfosalicylic acid (SSA) test result.

Multiple Myeloma - Bence Jones Protein (immunoglobulins)

List some false positives and false negatives for Protein dipstick test

False-positive
Highly buffered alkaline urine
Highly pigmented urine
High SG
Prolonged exposure of urine to strip pad
False-negatives
Proteins other than albumin
Microalbuminuria (low amount)

Briefly discuss the clinical significance of urine glucose testing.

Major screening test for diabetes mellitus and gestational diabetes

Explain why glycosuria occurs in the presence of hyperglycemia.

Blood glucose levels greater than the renal threshold for glucose produce glucose concentrations in the filtrate that exceed the maximum reabsorptive capacity of the tubules

Explain why the following people would exhibit hyperglycemia and glucosuria: a person with hyperthyroidism

Hyperthyroidism: The hormone thyroxin opposes the action of insulin by breaking down glycogen to glucose

Explain why the following people would exhibit hyperglycemia and glucosuria: a person under extreme stress

Extreme stress: epinephrine inhibits insulin secretion

How can glycosuria occur in the absence of hyperglycemia?

Renal tubular dysfunction

Explain the purpose of glucose oxidase and peroxidase in the reagent strip test for glucose

Glucose oxidase catalyzes a reaction between glucose and room air to form hydrogen peroxide, which is then broken down by peroxidase to produce oxygen that oxidizes a chromogen, producing a color

How is the dipstick glucose reported?

Neg, Trace, 1+, 2+, 3+, 4+
100mg/dl to 2 g/dL

List reasons for false negative and false positive reactions for glucose on dipstick

False Positive - Peroxide, oxidizing detergents used on lab equipment
False Negative- Ascorbic Acid and strong reducing agents, high ketone levels, High SG, and lowtemperature, Unpreserved specimen at Room temp is greatest error (bacteria)

Describe the copper reduction method for detection of urinary reducing substances, and discuss the current use of this procedure

Reduction of copper sulfate to cuprous oxide with alkali and heat
Color change: negative blue (CuSO4) through green, yellow, and orange/red (Cu2O)Only used to detect other reducing substance beside glucose in urine

List the urine dipstick test affected by Ascorbic Acid.

Blood
Bilirubin
Glucose
Nitrite
Leukocyte Esterase

What is the "pass through" phenomenon? How will failure to detect "pass through" affect Clinitest results?

Color passes through orange/red and returns to green/brown: rapid reaction and may miss the strongest reaction- seen with very high levels of reducing substance present

Which test is more sensitive for glucose?

Dipstick

List some interference reducing sugars on the Clinitest

Galactose
Lactose
Fructose
Maltose
Pentoses
Ascorbic Acid
Certain drug metabolites
Antibiotics

Which reducing substance other than glucose that is significant in newborns?

Galactose

Name the three "ketone bodies" appearing in urine

Acetone
Acetoacetic acid
B-Hydroxybutyrate

Name three causes of ketonuria.

Diabetes Mellitus
Vomiting
Starvation

What is the primary substance detected by sodium nitroprusside? Why is glycine added to the reaction?

Acetoacidic acid
Assumes the presence of B-hydroxybutyrare and acetone.

Differentiate between hematuria, hemoglobinuria, and myoglobinuria with regard to the appearance of urine and serum and clinical significance.

Hematuria- intact RBCs causing a cloudy urine
Hemoglobinuria-product of RBC destruction causing a clear red urine
Myoglobinuria-Heme containing protein in muscle tissue from muscle destruction causing clear,red/brown urine

Describe the chemical principle of the reagent strip method for blood testing, and list possible causes of interference.

Pseudoperoxidase activity of hemoglobin to catalyze a reaction between the heme component of both hemoglobin and myoglobin and the chromogen tetramethylbenzidine to produce an oxidized chromogen, which has a green-blue color

List possible causes of interference.

False positive - Menstrual contamination, strong oxidizing agents, vegetable peroxidase ,bacterial peroxidases
False negatives- >25 mg/dL Ascorbic acid, High SG, Formalin, Captopril, High Nitrites, Unmixed specimen

What is the significance of a speckled reaction on a blood pad?

Intact red blood cells (RBCs) are present.

Outline the steps in the degradation of hemoglobin to bilirubin to urobilinogen and finally urobilin

RBC→hemoglobin→Biliverdin→unconjugated bilirubin→conjugatedbilirubin→urobilinogen→stercobilinogen→urobilin

Discuss the principle of the reagent strip test for urinary bilirubin, including possible sources oferror.

Bilirubin glucuronide + Diazonium salt→azodye
False positives-urine pigments
False negatives-old specimens, >25 mg/dL ascorbic acid, increased nitrite

What is a good confirmatory test for a positive dipstick bilirubin and discuss the sensitivity of the test.

Ictotest tablet - 4x more sensitive than reagent strip. Interfering substances can be washed in the mat and bilirubin adheres to mat surface.

Describe the relationship of urinary bilirubin and urobilinogen to the diagnosis of bile duct obstruction, liver disease, and hemolytic disorders

Negative bilirubin indicates normal liver function. Increased Urobilinogen indicates excess bilirubin being processed in liver due to increased rbc destruction.
Increased bilirubin and increased urobilinogen indicates liver failure.
Increased bilirubin and normal to negative urobilinogen indicates bile blockage

Discuss the principle of the reagent strip test for urobilinogen for both Multistix and Chemstrip

Multistix - Ehrlich'sp-dimethylaminobenzaldehyde→red color in EU
Chemstrip-diazonium salt→red azadye in mg/dLmore specific

List possible sources of error for both Multistix and Chemstrip.

False positives-nonspecific reactions with porphobilinogen and other compounds (Multistix)
Not testing atroom temp, highly pigmented urine, higher after meals
False negatives -Improper preservation
High nitrite levels (Chemstrip)

Why should urobilinogen levels normally be detected in urine and discuss the significance when no urobilinogen is detected in urine?

Urobilinogen appears in the urine because as it circulates in the blood back to the liver, it passes through the kidney and is filtered by the glomerulus. Therefore, a small amount of urobilinogen is normally found in the urine.
If none is detected, suspect duct obstruction

What is the significance of a positive Nitrite urine test?

Rapid screening test for the presence of urinary tract infection (UTI)

Discuss the significance of chemical reaction of nitrate to nitrite

The chemical basis of the nitrite test is the ability of certain gram-negative bacteria to reduce nitrate, a normal constituent of urine, to nitrite, which normally does not appear in the urine.

Where the nitrate comes from in the urine.

Nitrate normally occurs in the urine from our diets of green vegetables.

Discuss the principle of the reagent strip test for nitrite

Para-arsanilic acid + Nitrite→Diazonium salt + tetrahydrobenzoquinolin→pink azodye

What are possible sources of error of the reagent strip test for nitrite

False negatives: Non-reductase bacteria present, insufficient contact time between bacteria in bladder and nitrate, lack of urinary nitrate, large quantities of bacteria converting nitrite to nitrogen, antibiotics, ascorbic acid, high SG
False positives: pigmented urine

What is the significance of a positive Leukocyte Esterase urine test?

Increased urinary leukocytes are indicators of UTI.

Which leukocytes are detected by the Leukocyte Esterase test?

Neutrophils
Eosinophils
Basophils
Monocytes

Does the degree of positivity of the tests correlate with the number of wbcs seen microscopically, Explain

The test is not designed to measure the concentration (number) of leukocytes but the esterase inside the granules, and the manufacturers recommend that quantitation of wbcs be done by microscopic examination

Describe the chemical principle of the reagent strip method for Leukocyte Esterase testing

The reagent strip reaction uses the action of LE to catalyze the hydrolysis of an acid ester embedded on the reagent pad to produce an aromatic compound and acid. Then the aromatic compound combines with a diazonium salt present on the pad to produce a purple azo dye.

List possible causes of interference for Leukocyte Esterase testing

False positives-strong oxidizing reagents, highly pigmented urine
False negatives-high concentrations of protein, glucose, oxalic acid, ascorbic acid, high SG, presence of certain antibiotics

What is the time limit before a leukocyte esterase result should be interpreted and why is this important?

2 minutes
Misinterpretation of results, possibly miss a faint reaction needing a longer time for reaction

Explain the principle of the chemical test for specific gravity.

The reagent strip reaction is based on the change in dissociation constant of a polyelectrolyte in an alkaline medium. The polyelectrolyte ionizes, releasing hydrogen ions in proportion to the number of ions in the solution. The higher the concentration of urine, the more hydrogen ions are released, thereby lowering the pH. Incorporation of the indicator bromothymol blue on the reagent pad measures the change in pH. As the specific gravity increases, the indicator changes from blue, through shades of green, to yellow. Readings can be made in 0.005 intervals by careful comparison with the color chart

Discuss any interference of testing for Specific Gravity on dipstick.

No interference from large molecules, urea and glucose, radiographic dye and plasma expanders
Slight elevation from protein
Decreased readings: urine pH 6.5 or higher

Compare reagent strip testing for urine specific gravity with osmolality and refractometer testing.

Reagent Strip: measures pKa of a polyelectrolyte in alkaline medium
Osmolality: measure of dissolved substances compared to known standards and colligativeproperties
Refractometer: measures refractive index of dissolved substances based on water