Biochemistry

AnalyzerPrepared By:

RahmatZaidDea

TEST FOR URINALYSIS: UroMeter 720 & SD UroColor

Purpose:Test for blood, bilirubin, urobilinogen, ketone, protein, nitrate, glucose, pH, spesific gravity, leucocytes & ascorbic acid in urine.

INTRODUCTION

SD Urocolor are plastic strips which are attached several separate reagent areas.

Test results may provide information regarding the status of carbohydrate metabolism, kidney and liver function, acid-base balance & urinary tract infection.

The results of a urine dipstick test may

point to a diagnosis of urinary tract

infection (UTI), kidney disease, diabetes, or

a urinary tract injury. If test results are

abnormal, other tests will be needed

before a definite diagnosis can be made.

Urine Dipstick Chemical Analysis

Urine dipstick is a narrow plastic strip which has several squares of different colors attached to it. Each small square represents a component of the test used to interpret urinalysis. The entire strip is dipped in the urine sample and color changes in each square are noted.

The color change takes place after several seconds to a few minutes from dipping the strip. If read too early or too long after the strip is dipped, the results may not be accurate.

Each color change on a particular square may indicate specific abnormalities in the urine sample caused by a certain chemical reaction.

The reference for color changes is posted on the plastic bottle container of the urine test strips. This makes for easy and quick interpretation of the urinalysis results by placing the strip next to the container and comparing its color changes to the reference provided.

PRINCIPLE

PRINCIPLE

Determine the intensity of different colors on the reagent strip test area. It does this

by irradiating the test area with light and detecting the refectance of different

wavelengths using an integrated sphere photo-detector.

This photo-detector is filtered to measure wavelengths of 550nm, 620nm, and

720nm using the integrated sphere.

720nm is the reference wavelength and the combination of 550nm and 620nm light

is used to differentiate between the different color combinations used in the urine

strips.

Results are calculated by a refection rate (R%) which is a percentage of the total

refectance of the testing wavelength and are printed automatically.

If the intensity of color on the strip is high then the refectance will be low conversely

if the intensity of the color is low then the refectance will be high.

Squares on the dipstick represent the following components in the urine:

specific gravity (concentration of urine)

acidity of the urine (pH)

protein in the urine (mainly albumin)

glucose (sugar)

ketones (products of fat metabolism)

blood, leukocyte esterase (suggestive of white blood cells in urine)

nitrite (suggestive of bacteria in urine)

bilirubin (possible liver disease or red blood cell breakdown)

urobilinogen (possible liver disease)

Testing Method

a) Dipsticks may be removed from the air tight, light sealed containers. It is important not to touch the reagent areas of the strip as this may alter test results.

b) Each reagent area should be immersed in urine by dipping.

c) The excess urine should be removed to prevent dilution of reagents or mixing of reagents between pads. This can be achieved by tilting the strip and allowing the urine to run off the edges. While blotting excess urine, ensure the chemicals from the different tests do not mix.

ACTIVE INGREDIENTS & CHEMICAL PRINCIPLES OF THE PROCEDURE

BLOODContains: Tetramethylbenzidine 12 mcg & Cumene Hydroperoxide 5 microliter.

Principle: This test is based on peroxidase like activity of hemoglobin, which catalyse the reaction of organic hydroperoxidase. Resulting color ranges from yellow to greenish blue.

When hemoglobin appears in urine, it indicates kidney disease or urinary tract infection (UTI).

Certain oxidizing contaminants such as hypochlorite may produce false positive results.

Microbial peroxidase associated with urinary tract infection may cause a false positive reaction.

BILIRUBIN

Test strip contains : Sodium Nitrate 13 mcg, Substitute aniline diazonium salt.

PRINCIPLE:Based on azo-coupling reaction of bilirubin with a diazonium salt in a acid medium to form an azodye. The resulting color ranges from white to dark pink.

Nitrate concentration of 0.1 mg% or greater may cause a false negative reaction.

UROBILINOGEN

Test strip contains: 4-Diethylaminobenzaldehyde 26 mcg.

PRINCIPLE:4-Diethylaminobenzaldehyde reacts with urobilinogen in a strong acid medium to produce pink color.

Formaldehyde concentration of 1%(v/v) may cause a false negative reaction.

KETONETest strip contains: Sodium Nitriprusside 0.19 mg

PRINCIPLE:Nitroprusside react with acetoacetic acid.

False positive results may occur with highly pigmented urine specimens.

PROTEINTest strip contains: Tetrabromophenol blue 2 mcg.

PRINCIPLE:When pH is held constant by a buffer, indicator dyes release H+ ions because of protein presence.

False positive results may be found in strongly basic urine.

NITRITETest strip contains: P-arsanilic Acid 56 µg & 1,2,3,4-Tetrahydrobenzoquinolin-3-ol 10 µg.

PRINCIPLE:Based on diazotization reaction of nitrite with an aromatic amine to produce a diazonium salt. Then, followed by an azo-coupling reaction of this diazonium salt with an aromatic compound on the reaction pad. The azo dye produced causes a color change from white to pink.

The reaction reveals the presence of nitrite and hence indirectly of nitrite-forming bacteria in the urine.

GLUCOSETest strip contains: Glucose Oxidase 1.1 unit, Peroxidase 0.13 unit, Potassium Iodide

PRINCIPLE:Based on the specific glucose-oxidase / peroxidase reaction.

Certain oxidizing contaminants such as hypochlorite may produce false positive results.

pHTest strip contains: Methyl Red 1.3 µg , Bromothymol Blue 9 µg.

PRINCIPLE:Measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at 25 °C (77 °F). Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline.

pH of urine is an important indicator of certain metabolic, kidney, gastrointestinal and respiratory factors.

SPECIFIC GRAVITYTest strip contain: Bromothymol Blue 18 µg.

PRINCIPLE:Reflect the ion concentration of urine correlates well with the refractometric method. In the presence of cations, protons are released by a complexing agent & produce a color change in the indicator bromothymol blue from blue via blue-green to yellow.

LEUCOCYTETest strip contains: DERIVATIZED Phenylpyrrole 9 µg , Diazonium salt 7 µg.

PRINCIPLE:Granulocytic leucocytes contain asterases that catalyze the hydrolysis of the derivatized pyrrole amino acid ester to liberate 3-hydroxy-5-phenyl pyrrole then reacts with a diazonium salt to produce red-purple product.

ASCORBIC ACIDTest strip contain: 2,6-Dichlorophenol Indophenol 0.75 mg.

PRINCIPLE:This test is based on reduction / oxidation reaction of ascorbic acid with thiazine, oxazine compounds.

False positive result may be found in strongly basic urine.

Normal urine is typically light yellow and clear without any cloudiness. Obvious abnormalities in the color, clarity and cloudiness may suggest possibility of:

An infection (cloudy urine)

Dehydration (dark urine color)

Red blood in the urine also referred to as hematuria (red urine

color)

Liver disease (urine the color of tea)

Breakdown of muscle (orange or tea colored urine)

Certain medications may also change the color of urine. Very

foamy urine may represent large amounts of protein in the urine

(proteinuria).

Interpretations of Urinalysis

Urine pHThe pH of urine is another one of the urinalysis values that can help doctors determine if a medical problem exists. Normal urine pH ranges from 5.0 to 9.0. Any urinalysis values outside of that range can indicate a problem with acidity or alkalinity. If the PH level of the urine is low, the urinalysis values for nitrates can be affected. Low pH can lead to a false negative for nitrites, which means that nitrites won't show up on the urinalysis even if they are present.

NitritesNitrite levels are also tested with the urinalysis. These values can be elevated when bacteria are found in the urinary tract. However, urinalysis results for nitrites can be affected by many issues. False negatives can be caused by other infections, lack of available nitrates, and a quick conversion of nitrites to nitrogen. As mentioned above, low pH can also cause a false negative.

ProteinProtein is one of the many urinalysis values that can help doctors

determine if there is a problem with the kidneys or the bladder.

When the kidneys are damaged, many substances can leak from the

kidneys into the urine. Since proteins are made of very large

molecules, the presence of protein the urine can indicate advancing

kidney problems.

CastsIf urinalysis results indicate that there are casts in the urine, this can

indicate several issues. Casts appear when cells collect in the distal

tubules and become concretions. The color of the casts can help

doctors determine what problem exists. Red casts indicate nephritic

syndrome, white casts indicate kidney infection and muddy brown

casts can indicate renal failure.

•Refrigerated urine sample not returned to room temperature prior to testing

•Urine contaminated with disinfectant – from skin or cleaning prior to

collection

•Expired reagent strips

•Improper storage with exposure to air or light

•Leakage of reagent chemical from one test to another if the test is read

vertically rather than horizontally

•Tests read at inappropriate times

•Highly pigmented urine

•Failure to use control urine to check accuracy of strip

POTENTIAL CAUSES OF ERROR

Parameter Expected Results Interpretation of Results Causes of False Positives (or Increase)

Causes of False Negatives (or Decrease)

Specific Gravity 1.005 to 1.065 moderate to high levels of protein

alkaline urine

pH 5.0 to 6.0 Acidic: meat diet; acidosis; low chloride; acidifying agents Alkaline: vegetable based diet; bacterial infection; alkalosis; urine exposed to air for extended times; administration of alkalinizing agents; postprandial tide

glucose in urine

Glucose Negative to Trace Positive: Chronic or transient hyperglycemia; post administration of certain drugs; rarely-fanconi-like syndrome

hydrogen peroxide; bleach ascorbic acid; ketones; increased specific gravity; cold urine; expired reagent strips

Protein Negative to Trace hemorrhage; urinary infection; intravascular hemolysis; renal disease

alkaline urine; disinfectant residue

dilute or acidic urine

Blood Negative Positive: hematuria: trauma, infection, inflammation, infarction, calculi, neoplasia, or coagulopathy hemoglobinuria: myoglobinuria

bleach contamination; high levels of bromide or iodide; bitch in heat

poorly mixed urine

Bilirubin Negative Positive: can be normal in dogs; ALWAYS abnormal in cats; indicates liver disease, bile duct obstruction, starvation; hemolysis; pyrexia

high doses of chlorpromazine; etodolac metabolites

ascorbic acid; nitrates

SUMMARY