GROUP 1

OBJECTIVES:

To discuss the principle behind every

test for urine
To discuss the results and
implications
To name the different diseases
associated with every test
 Definition of term:

 by- product or fluid secreted

by the kidneys, transported by
the ureter to the urinary
bladder and voided through
urethra
 Urine production is produced by the
process of:
1.Filtration
2.Reabsorption
3.Tubular secretion
 a. General characteristics:
COLOR CAUSE CLINICAL CORRELATIONS
Colorless recent fluid commonly observed
consumption with random specimen
Yellow presence of urochrome -----
Pale yellow  Polyuria or diabetes  increased 24- hour
insipidus volume
 Diabetes mellitus  elevated SG and
 Dilute random positive glucose test
specimen result
 recent fluid
consumption
Dark yellow concentrated specimen may be normal after
strenuous exercise or in
the first morning
specimen
Amber ----- dehydration from fever
and burns
• RED COLOR URINE
• - with RBC’s
• - hematuria
• - hemoglobinuria
• - myoglobinuria
 ODOR CAUSE
Aromatic normal
Foul, ammonia- like bacterial decomposition
Fruity, sweet ketones (diabetes mellitus,
starvation, vomiting)
Maple syrup maple syrup urine disease
Mousy phenylketonuria
Rancid tyrosinemia
Sweaty feet isovaleric acidemia
Cabbage methionine malabsoprtion
Bleach contamination
TRANSPARENCY
Clear
Hazy
Cloudy
Turbid
Milky
 Reagent strip
provide a simple, rapid means for
performing medically significant
chemical analysis of urine.
consist of a chemical- impregnated
absorbent pads attach to a plastic
strips.
 Reagent strip technique
1. Dip the reagent strip briefly into a well- mixed uncentrifuged
urine specimen at room temperature.
2. Remove excess urine by touching the edge of the strip to the
container as the strip is withdrawn.
3. Blot the edge of the strip on a disposable absorbent pad.
4. Wait the specified amount of time for the reaction to occur.
5. Compare the color reaction of the strip pads to the
manufacturer’s color chart in good lighting.
10- parameter reagent strip
10- parameter reagent strip
 LEUKOCYTES
Clinical significance:

Principle:
 Granulocytic leukocytes  Bacterial and nonbacterial UTI.
contain esterases that  Inflammation of the urinary
catalyze the hydrolysis of tract.
the derivatized pyrrole  Screening of urine culture
amino acid ester to specimens.
liberate 3-hydroxy-5-phenyl
pyrrole. This pyrrole then
reacts with a diazonium
salt to produce a purple
product.
 nitrite
Clinical significance:
 This test depends upon the
Principle:
conversion of nitrate to nitrite
by the action of Gram  Cystitis
negative bacteria in the urine.  Pyelonephritis
 At the acid pH of the reagent  Evaluation of antibiotic therapy
area, nitrite in the urine  Monitoring of patients at high risk
reacts with p-arsanilic acid to for UTI
form a diazonium compound  Screening of urine culture
in turn couples with 1,2,3,4- specimens
tetrahydrobenzo(h)quinolin-3-
ol to produce a pink color.
 urobilinogen
Clinical significance:
 This test is based on a
Principle:
modified Ehrlich reaction,
 Early detection of liver disease
in which p-
 Liver disorders, hepatitis, cirrhosis,
diethylaminobenzaldehyde
carcinoma
in conjunction with a color
 Hemolytic disorder
enhancer reacts with
urobilinogen in a strongly
acid medium to produce a
pink-red color.
 PRotein
Clinical significance:
 This test is based on the
Principle:
protein-error-of-indicators
 Pre-renal
principle. At a constant pH,
 Renal
the development of any
green color is due to the  Post-renal
presence of protein.  Tubular disorders
 PH
Clinical significance:
 The test is based on the
Principle:  Respiratory or metabolic
double indicator principle acidosis/ketosis
that gives a broad range of  Respiratory or metabolic alkalosis
colors covering the entire  Defects in renal tubular secretion
urinary pH range. Colors  Reabsorption of acids and bases-
range from orange through renal tubular acidosis
yellow and green to blue.  Renal calculi formation
 Treatment of UTI
 Precipitation/ identification of
crystals
 Determination of unsatisfactory
specimens.
 blood
Clinical significance:
 Chemical tests for blood
Principle:  Hematuria
use the pseudoperoxidase  Hemoglobinuria
activity of hemoglobin to  Myoglobinuria
catalyze a reaction between
hydrogen peroxide and the
chromogen
tetramethylbenzidine to
produce an oxidized
chromogen which has a
green-blue color.
 HEMATURIA HEMOGLOBINURIA MYOGLOBINURIA
1. Renal calculi 1. Transfusion 1. Muscular trauma/
reactions crush syndromes
2. Glomerulonephritis 2. Hemolytic anemias 2. Prolonged coma
3. Pyelonephritis 3. Severe burns 3. Convulsions
4. Tumors 4. infections/ malaria 4. Muscle- wasting
disease
5. Trauma 5. Strenuous exercise/ 5. alcoholism/
red blood cell trauma overdose
6. Exposure to toxic 6. Brown recluse 6. Drug abuse
chemicals spider bites
7. Anticoagulants 7. Extensive exertion
8. Strenous exercise 8. cholesterol-
lowering statin
medication
 Specific gravity
Clinical significance:
 This test is based on the
Principle:  Monitoring patient hydration and
apparent pKa change of dehydration
certain pretreated  Loss of renal tubular concentrating
polyelectrolytes in relation ability
to ionic concentration. In  Diabetes insipidus
the presence of an  Determination of unsatisfactory
indicator, colors range from specimens due to lower
deep blue-green through concentration
green and yellow-green in
urines.
 ketone
Clinical significance:
 This test is based on the
Principle:  Diabetic acidosis
development of colors  Insulin dosage monitoring
ranging from buff-pink to  Starvation
purple when acetoacetic  Malabsorption/ pancreatic disorders
acid reacts with  Strenuous exercise
nitroprusside.  Inborn errors of amino acid
metabolism
 Vomiting
 bilirubin
Clinical significance:
 This test is based on the
Principle:  Hepatitis
coupling of bilirubin with  Cirrhosis
diazotized dichloraniline in a  Other liver disorders
strongly acid medium. The  Biliary obstruction (gallstone,
color ranges through carcinoma)
various shades of tan.
 glucose
Clinical significance:
 This test is based on a
Principle:  Hyperglycemia- associated
double sequential enzyme  Renal- associated
reaction.
 Reagent strips employ the
glucose oxidase testing
method by impregnating
the testing are with a
mixture of glucose oxidase,
peroxidase chromogen and
buffer.
 HYPERGLYCEMIA- RENAL- ASSOCIATED
ASSOCIATED
Diabetes mellitus Fanconi syndrome
Pancreatitis Advance renal disease
Pancreatic cancer Osteomalacia
Acromegaly Pregnancy
Cushing syndrome
Hyperthyroidism
Pheochromocytoma
CNS damage
Stress
 DIPSTICK PRINCIPLE INVOLVE REAGENT
Leukocyte Granulocytic esterase •Indoxyl carbonic acid
reaction •Diazonium salt
Nitrite Greiss reaction •p-Arsalinic acid
Urobilinogen Modified Ehrlich’s reagent •p-dimethylamino
benzaldehyde
Protein Protein error of indicators •Tetrabromophenol blue
pH Double indicator system •Bromthymol blue
•Methyl red
 DISPTICK PRINCIPLE INVOLVE REAGENT
Blood Pseudoperoxidase •Tetramethylbenzidine
activity of hemoglobin •Buffered organic
peroxide
SG pKa change of a •Bromothymol blue
polyelectrolyte •Poly (methyl vinyl
ether-alt-maleic
anhydride)
Ketone Sodium nitroprusside •Sodium nitroprusside
reaction in a strongly
basic medium
Bilirubin Diazo reaction •2,4- dichloroaniline
diazonium salt
Glucose Double sequential •Glucose oxidase
enzyme reaction •Peroxidase
•Potassium iodine
 b. Isolation of urea
 Urea
- uses as a fertilizer and food supplement, as well as a starting
material for the manufacture of plastics and drugs.
- colorless, crystalline substance that melts at 132.7 degrees celsius
and decomposes before boiling.
- chief nitrogenous end product of the metabolic breakdown of
proteins.

 Reageant: Baryta mixture

 c. Test for urea formation of biuret
 Principle :
 Urea when heated
decomposes with the
liberation of ammonia and
the formation of biuret.
Biuret is dissolved in water
and develops a violet color
forming a complex with
alkaline copper sulphate
solution.
 Expected result:
 violet color complex
 d. Isolation of uric acid
 Uric acid is a chemical produced when your body breaks down foods that
contain organic compounds called purines.

 Most uric acid is dissolved in the blood, filtered through the kidneys, and
expelled in the urine.

 Normal Uric acid levels are 2.4-6.0 mg/dL (female) and 3.4-7.0 mg/dL
(male).

 Hyperuricemia is the name of the disorder that occurs when you have too
much uric acid in your body.
• Diseases associated with high uric acid:
 Gout – most common disease
 Diabetes
 Leukemia
 Hypoparathyroidism
 Acute kidney failure
 Multiple myeloma
 Metastasized cancer
• Disease associated with low uric acid:

 Liver or kidney disease

 Fanconi syndrome

 Wilson’s disease
 e. test for uric acid (murexide test)

 Principle:
 Murexide test for uric acid
is a specific test for the
determination of uric
acid in blood and urine.
This test is mainly used in
order to help diagnose
gout.
 Expected Result:
 reddish-violet
 f. Creatinine (nitroprusside test)
 Creatinine
- chemical waste product that’s normally filtered out of your blood by
your kidneys.
 Creatinine urine test
- evaluates how well your kidneys are working by measuring the
amount of creatinine in your urine
 Nitroprusside test (Wey’s test)
- chemical test used for detecting the presence of free thiol groups
of cysteine in proteins
- proteins with the free thiol group give a red colour when added
to sodium nitroprusside with ammonium hydroxide.
- Sodium Hydroxide (NaOH) is used to make the solution alkaline.

- Expected Result: RED COLOR

g. Inorganic physiological
constituents:

-phosphates
-iron
-sulfates
-chlorides
 g. Inorganic physiological constituents: PHOSPHATES

 A simple qualitative method to determine the presence of phosphate ions in

a sample. A small amount of the sample is acidified with concentrated nitric
acid to which a little ammonium molybdate is added.
 The presence of phosphate ions is indicated by the formation of a bright
yellow precipitate layer of ammonium phosphomolybdate.
 A test to measure phosphate in urine may be done to:
• 1. Help diagnose kidney problems that affect phosphate levels.
• 2. Help find the cause of kidney stones.
 g. Inorganic physiological constituents: iron

 These tests are used together to detect and help diagnose iron deficiency
or iron overload. In people with anemia, these tests can help determine
whether the condition is due to iron deficiency or another cause, such
as chronic blood loss or some other illness.

 Hemoglobin (iron-containing pigment in red blood cells; may indicate injury

of the urinary tract, anemia, or infection)
 g. Inorganic physiological constituents: Sulfates

 Reagent: Barium Chloride (BaCl2), Nitric Acid (HNO3)

 Expected Result: Formation of white precipitate

 Principle: Urinary Sulphate is precipitated as barium sulphate upon reacting

with barium chloride solution

 Interpretation:Increase and Decrease on the concentration of Sulfates could

cause various types of diseases
 g. Inorganic physiological constituents: chlorides

 Precipitating Agents: Nitric Acid(HNO3) and Silver Nitrate(AgNO3)

 Positive Result: Formation of a white precipitate

 Principle: Silver chloride is precipitated in the presence of nitric acid and

silver nitrate

 Interpretation: Increase and decrease on the concentration of Chlorides

could cause various types of diseases.
h. Pathological constituents

-glucose benedict’s test

-albumin (heller’s ring test)
-bile pigments (gmelin’s test)
-bile acids (petenkofer’s test)
-acetone test (nitroprusside test)
ABNORMAL/PATHOLOGICAL
URINE

A Urine that contains Substances which are not

present in easily detectable amounts in urine of a
normal healthy individual but are present in the
urine under certain diseased person and are said to
be “Abnormal or Pathological” constituents of Urine.
 Abnormal Constituents of Urine

• Reducing Sugars
• Ketone Bodies
• Proteins
• Blood
• Bile Salt
• Bile pigments
• Urobilinogen
 h. Pathological constituents: glucose benedict’s test

 Principle: Benedict’s Reagent contains sodium carbonate, copper sulphate

and sodium citrate. In the alkaline medium, provided by sodium carbonate,
the copper remains as cupric hydroxide. Sodium citrate acts as a stabilizing
agent to prevent the precipitation of cupric hydroxide. In alkaline medium,
sugars form enediols, cupric ions are reduced, and corresponding sugar is
oxidized to sugar acid.

 Reagent Used: Benedict’s Reagent

 Results: A Positive Benedict’s Test signifies Glycosuria

• Benedict’s Test is a semi quantitative test. The color of the
precipitate gives a rough estimate of the reducing sugars
present in the given sample:
• Green Color : up to 0.5g %
• Green Precipitate :0.5 – 1.0g % (+)
• Yellow Precipitate: 1.0 – 1.5g % (++)
• Orange Precipitate : 1.5 – 2.0g % (+++)
• Brick Red Precipitate: >2.0g % (++++)
 h. Pathological constituents: albumin (heller’s ring test)

 Principle: Concentrated HNO3 causes denaturation and hence precipitation

of proteins

 Precipitating Agent: Nitric Acid (HNO3)

 Positive Result: Precipitate is formed

 h. Pathological constituents : bile pigment (Gmelin’s test)

 Gmelin’s test is a specific test used for the qualitative detection of

bile pigments in urine. The bile pigments bilirubin and biliverdin are
almost entirely excreted through the bile by the liver but in
conditions like obstructive jaundice, where bile ducts get blocked,
the bile pigments start appearing in the urine.

 Principle: Nitric acid oxidizes bilirubin to biliverdin giving different

colors from green to violent.

 Reagent: Nitric acid (HNO3).

 h. Pathological constituents: bile acids (petenkofer’s test)

 This test is used to prove the presence of bile salt.

 Principle: Bile salt will reacts with hydroxymethylenephurphural to

forms red solution. Hydroxymethylenephurphural is formed of sugar
that dehydrated with sulfuric acid.

 Reagent: H2SO4
 h. Pathological constituents: acetone test (nitroprusside test)

 A chemical test used for detecting the presence of free thiol groups
of cysteine in proteins.
 The nitroprusside reaction is usually used for the identification of
ketones in urine testing.

 Principle: Proteins with the free thiol group give a red color when
added to sodium nitroprusside with ammonium hydroxide. Some
proteins test positive when denatured, indicating that thiol groups
are liberated.

 Reagent: Alkalinized with NaOH

 RESULTS: (physical)

Normal PATHOLOGICAL

PARAMETER OBSERVATION PARAMETER OBSERVATION

Color Light yellow/ Color Light yellow/
pale yellow pale yellow
Transparency Clear Transparency Clear
 Results: (chemical)
PARAMETER OBSERVATION
Urobilinogen 0.1
Glucose -
Bilirubin -
Ketones -
SG 1.020
Blood -
pH 6.5
Protein -
Nitrite -
Leukocytes -
 Results: (chemical)
PARAMETER OBSERVATION
Urobilinogen 0.1
Glucose ±100
Bilirubin -
Ketones -
SG 1.020
Blood -
pH 6.5
Protein -
Nitrite -
Leukocytes -
 How did you identify the physiologic and
pathologic constituents of the urine?

 Examining the physical/macroscopic appearance of urine

which includes its color, transparency and for the
identification of the pathological constituents of urine we
performed the chemical examination which is the reagent
strip test to detect the pH, SG, glucose, protein, ketones,
blood, bilirubin, uribilinogen, leukocytes and nitrate in our
urine sample.