CHAPTER FOUR

Chemical analysis of urine

Acknowledgements

 Addisa Ababa University

 Jimma University
 Hawassa University
 Haramaya University
 University of Gondar
 American Society for Clinical Pathology
 Center for Disease Control and Prevention-Ethiopia
 Objective:

 At the end of this chapter the students will able:

 to carry out how to perform chemical tests and show
the various types of methods used to determine the
chemical constituent.
 Know the interfering factors during there work
 Expected to differentiate the principles of each test
 Explain the test procedures
Chapter outline
 Determination of Urinary Ph
 Determination of Urinary sugar
 Determination of Urinary keton bodies
 Determination of Urinary protein
 Determination of Urinary hemoglobin
 Determination of Urinary Bilirubin
 Determination of Urinary urobilinogen
 Determination of Urinary urobilin
 Determination of Urinary Porphybilinogen
 Determination of Urinary lecuocyte estrase
 Determination of Urinary Nitrite
Chapter outline cont’d

 Determination of Urinary Calcium

 Determination of Urinary Urinary indicant
 Determination of Urinary Melanin
 Determination of Urinary Ascorbic Acid
 Determination of Urinary phenyl pyruvic Acid
 Determination of Urinary Mucopolysacchride
 Determination of Urinary Hemosiderine
 Introduction
 Chemical analysis of urine deals with those procedures
or examinations which are accomplished by means of
chemical reactions. .
 Chemical test are done for a quick screening of urine for
certain substances which have a diagnostic significance.
 In this category of tests, abnormal or pathological
dissolved constituents of urine (chemicals) performed in
routine urinalysis
 Introduction Cont…

 Chemical analysis of urine is important in the detection

of many diseases.
 Urine contains normal chemical compositions. But in
abnormal ( pathological ) conditions its composition
varies in kind and quantities.
 So the chemical changes of urine can indicate
disease at the early stage.
 Introduction Cont…

 Urine composiotion depends greatly on how much

and what specific waste material is to be excreted.
 Urea, creatinine, uric acid, ammonium salts,
chlorides, sulphates and phosphates of sodium,
potassium, calcium and magnesiumn are the normal
composition of urine.
 Glucose, protein, ketone bodies, bilirubin , bile salts etc.
are the abnormal constituents of urine. Normaly these
substances do not appear in the urine in detectable
amount . So their appearance in the urine shows the
pathological condition.
Chemical analysis

 Chemical analysis of urine can be:

 Screening test
 Qualitative test
 Quantitative
 Chemical analysis

 Screening test : tells the presence or absence of the

abnormal components of urine
 Qualitative test: tells semi-quantitatively the amount of
solutes that exist at the detectable level in pathological
or physiological conditions
 Quantitative test: tells the exact amount of abnormal
constituents if they are detectable
PH
 Along with the lungs, the kidneys are the major
regulators of the acid-base content in the body. They do
this through the secretion of hydrogen in the form of
ammonium ions, hydrogen phosphate, and weak
organic acids, and by the reabsorption of bicarbonate
from the filtrate in the convoluted tubules
 A healthy individual usually produces a first morning
specimen with a slightly acidic pH of 5.0 to 6.0; a more
alkaline pH is found following meals (alkaline tide). The
pH of normal random samples can range from 4.5 to
8.0.
 PH

 pH is the unit that describes the acidity or alkalinity of a

solution
 In chemical terms, acidity refers to the hydronium ion
(H3O+) concentration of a solution
 and alkalinity refers to its hydroxyl ion (OH-)
concentration.
 These concentrations are usually expressed in terms of
pH.
pH
 Because the kidney is generally working to eliminate
excess acid, the pH of the urine is normally between 5
and 6.
 The kidney is capable of producing urine ranging in pH
from 4.5-8.0. Although the kidney is essential in
controlling the pH of blood and extracellular fluid,
measurement of urinary pH are not necessarily used to
obtain information about this role.
Reasons for measurement of urinary pH

 Alkalinity of freshly voided urine, indicate a urinary tract

infection.
 The urinary pH helps in the identification of crystals of
certain chemical compounds
 Persistently acidic urine in diabetic acidosis resulting
from an accumulation of ketone bodies.
 To control management of kidney infections, in cases
of renal calculi (stones) and during the administration of
certain drugs.
Method of measuring urinary pH

 Litmus paper
 Nitrazine paper
 reagent strip
 Glass electrode (pH meter
 Litmus paper
 In this technique pH measurement takes place by using
blue, and red litmus paper.
 Procedure
 Collect a freshly voided well mixed urine
 Tear small blue litmus paper and red litmus paper
 Dip the paper in the urine and remove immediately
 Look for color change of blue litmus paper and red
litmus paper.
 Interpretation:
 If blue colors of paper changes to red, it indicates the
acidity of the urine. If the red litmus paper change to
blue, it indicates that the urine is alkaline.
 Nitrazine paper

 The paper is impregnated with sodium dinitro

phenolazo – naphthal disulphonate chemical.
 a paper that changes its color from yellow (for acidic
urine) to blue (for alkaline urine).
 Unlike litmus paper, the color change is matched
with reference color chart.
Procedure
 Tear small nitrazine paper
 Dip the paper in freshly voided urine sample and remove
immediately
 Compare the color change with that of reference color
chart.
 Record the value of color change form reference color
chart.
 Note: Most nitrazine paper is manufactured with color
charts ranging from pH 3 or 4 to pH 9. However,
nitrazine paper has an accurate range of only 4.5-7.5.
 Reagent Strip Test

 Principle
 Utilize a methyl red and bromthymol blue double
indicator system that measures urine pH from 5 to 9.
 The Methyl red is used to indicate a pH range from
4.4 to 6.2 with a color change from red to yellow
 Bromthymol blue indicates a pH change from 6.0 to
7.6 as seen by a color change from yellow to blue.
Chemical Exam

 When the test strip is

dipped in urine the
reagents are activated
and a chemical
reaction occurs.
 The chemical reaction
results in a specific
color change.
Procedure
 Dip the test – strip in the urine specimen. Remove the
test-strip immediately and let the excess urine drain off
on a paper towel, or tap the edge of the strip.
 Read the color change
 Report the result according to the color chart provided
by manufacturer.
 Always read the test strip in good white light and
ignore color developing on the test area after the
period specified as the reading time of the test.
 Be careful not to wet the reagent strip excessively.
So that the acid buffer from the protein area runs into
the pH area, causing an orange discoloration.
Elevated pH (alkaline)

(Normal for comparison)

Interpretation and limitation

 Interpretation
 Report the exact color change of the stripe after
dipping it in the urine sample
 Samples should not be wait more than 30.
 Limitation
 Growth of bacteria if there is delay
 Contamination of the urine container with blenches
 Reading of results out of the limitation period
General Precaution for reagent strips
 Store strips in a cool, dry place to prevent deterioration
 Do not use strips if they are discolored
 Do not touch the test areas
 Keep the test areas away from detergents or other
contaminating substances
 Dip the test areas into the urine specimen completely,
but briefly to avoid dissolving the reagents out of the test
area.
General Precaution for reagent strips …

 Remove only the number of reagent strips required at

a particular time, and keep the container tightly
covered at all times to prevent deterioration
 Read the test results carefully, at the time specified by
the manufacturer, in a good light and with the test
area held near the appropriate color chart on the
bottle label
pH meter
 Hydrogen ion activity can be
measured with a pH meter
calibrated to this scale of 0-
14 with standard solutions.
 The meter measures
potential of hydrogen ion
activity or pH of an aqueous
solution.
pH Meter

 The pH
measurement
system consists of 2
electrodes:
a measuring
electrode capable of
detecting hydrogen
ions and
 the reference
electrode
pH measurement

 The pH electrode is a glass electrode, which

develops a voltage potential difference when the
pH of the sample differs from the pH of the
electrode fill solution, saturated KCl.
pH measurement

 The reference electrode is

 silver/silver chloride electrode with a constant voltage
 filled with saturated KCl
 separated from the sample by a cellulose membrane.
Clinical Significance
As indicated, one of the functions of renal system is
to regulate pH of blood i.e. keep pH of blood at 7.4
+ 0.05.
This is done by absorption or release of hydrogen
ion, especially at distal convoluted tubules of the
nephron, depending on the pH of blood, i.e.
hydrogen ion absorbed from surrounding blood
capillaries of nephrone when pH is acidic (below
7.35), and release from nephron to the surrounding
blood vessels when pH of blood is alkaline (above
7.45).
Clinical Significance cont’d…

 Persistent alkaline urine (pH > 6)

may be caused by:
UTI
Renal failure
Vomiting
Anorexia nervosa
metabolic Alkalosis.
Alkalizing drugs such as streptomycin, kanamycin etc.
certain vegetables, like citrus fruits.
milk productes
Clinical Significance cont’d…
Persistent acid urine (pH < 6)
may be caused by:
 Diarrhea
 Malabsorption syndromes
 Diabetic ketoacidosis
 Dehydration
 Fever
 Starvation
 And also certain drugs such as – Phenacetic
 high protein diet may also result in acidic urine, but
this is not a pathological condition.
Causes of Acid and Alkaline Urine
Determination of Glucose

 Glucose is the sugar most commonly found in the urine,

although other sugars , such as lactose, fructose ,
galactose, and pentose, may be found under certain
condition.
 The presence of detectable amount of glucose in the
urine is known as glycosuria.
 Normally almost all the glucose, which passes from the blood
into the glomerular filtrate, is reabsorbed back into the
circulation by the kidney tubules( proximal convoluted
tubules ).
 Usually less than 15 - 20 mg/dl (0.8 mmol) is excreted in the
urine. But this amount cannot be detected by the routine
laboratory tests.
Glucose cont’d…

 The blood glucose concentration normally lies between 65

and 110 mg/dl.
 After a meal it may increase to 120 - 160 mg/dl.
 If the blood glucose concentration becomes too high
(usually greater than 170 - 180 mg/dl), the excess glucose
will not be reabsorbed into the blood and glucose start
appearing in urine.
 The lowest blood glucose concentration that will result in
glycosuria is termed as the renal threshold.
 The most common condition in which the renal threshold
for glucose exceeds is diabetes mellitus
 Causes of Glycosuria

 Physiological
 Pathological

 Physiological
a. After large ingestion of carbohydrates
b. Anything that stimulates sympathetic nervous system
c. 15 to 20% cases of pregnancy
d. Renal Glycosuria:
Causes of Glycosuria
Pathological Glycosuria
A. Diabetes mellitus
 Diabetes Mellitus is a chronic polygenic syndrome with
impaired carbohydrate metabolism.
 Diabetes mellitus and its complications (diabetic
ketoacidosis and nonketotic hyperosmolar syndrome)
are the most common disorders of carbohydrate
metabolism.
Metabolic changes in diabetes mellitus
Causes of Glycosuria

B. Glycosuria due to other endocrine disorders

 A number of endocrine disorders can cause

hyperglycemia, and this may result in glycosuria,
e.g. - Hyperthyroidism
- Hyperadrenalism
- Hyperpitutarism
- Some diseases of pancreas
Causes of Glycosuria cont’d
 The carbohydrate metabolism is impaired due to
deficiency or ineffectiveness of insulin (peripheral insulin
resistance) or decreased insulin/anti-insulin hormone
ratio leading to chronic hyperglycemia and glycosuria
along with secondary changes in metabolism of protein,
lipids, water and electrolytes with grave consequences, if
not treated.
 Typically, its symptoms include polydypsia (excessive
thirst), polyuria (increased frequency of urination),
polyphagia (hunger), glucosuria
Types of Urinary Glucose Test

 Non specific
reduction tests based on the reduction of certain metal
ion by glucose
 Specific
Enzymatic tests based on the action of glucose oxidase
on glucose
Non-Specific Test for Glucose

 glucose is acting as a reducing agent and any compound

with a free aldehyde or ketone group will give the same
reaction.
 Non-Specific Hence glucose is not the only reducing
substance that may be found in urine.
 Urine contains non-glucose reducing substances
(NGRS) such as: uric acid, creatinine, galactose,
fructose, lactose, pentose, homogenestic acid, ascorbic
acid chloroform, and formaldehyde
Non-Specific Test for Glucose

 Commonly used non-specific tests for urinary sugar are:

-Benedict’s Qualitative test

-Fehling test

-Clinitest tablet test

Benedict’s Qualitative Test
Principle
 When urine is boiled in an alkaline copper sulphate
solution, glucose and other reducing substances reduce
(convert) the blue copper (II) in Benedict's qualitative
reagent to copper (I) oxide (Cu2O), which is orange to
red in color.
 A positive reaction is graded as a change in color
ranging from blue to green, yellow, orange and finally red
The overall reaction

 The copper (II) ions are supplied in Benedict's qualitative

reagent in the form of copper sulphate (CuS04).
 In the presence of a strong alkali this is converted to
copper ( I) oxide (Cu2O ).
 The heat is supplied by means of a boiling-water (100 OC)
bath. The tubes are brought back to room temperature,
and the results are read when convenient
procedure
1. Arrange test tubes (12ml)in a row rack.
2. Take two extra tubes for control runs
negative with water and
positive with 1.0 g/dl of glucose solution.
3. Add eight drops of well-mixed urine
4. Add 5ml of Benedict’s reagent.
5. Mix well and place in a boiling water bath for 5 min
6. Remove and cool to room temperature .
7. A positive : presence of a fine yellow to brick red
8. Graded on the basis of the color of solution.
Grade results according to the following criteria:

 Negative: No change in the blue color of the reagent or

the occurrence of a white or green precipitate from
phosphates in the urine.
 Trace: Slight amount of yellow precipitate with a
greenish blue to bluish
 green mixed solution. (This represents less than
500mg/dl of sugar).
 + : Moderate amount of yellow precipitate often referred to as
apple green. (Approximately 500mg/dl of sugar).
 ++: Large amount of yellow precipitate with a yellowish green,
often called muddy  green mixed solution. (Appr. 750mg/dl of
sugar).
 Fehling’s Test

 Principle: The same as benedict test

Fehling’s reagent

Solution A
Copper sulphate 34.65 g
Distilled water 500 ml
Solution B
Sodium hydroxide 125 gm
Sodium potassium
tartarate173 gm
Distilled water 500 ml
Procedure

 Take equal volume of Fehling’s solution A and solution B.

 Mix and boil.
 Add a few drops of urine and boil again.
 If sugar is present, the same type of change occur as in
the Benedict’s test and are reported similarly.
Advantage of Bendict’s Test over Fehling’s test

- It uses a single solution already prepared

- It uses a more stable reagent
- It is about ten times more sensitive and traces of glucose
will not be caramelized
- If the test is carried out using the proper proportions of the
reagent and urine, it is more specific, as uric acid and
creatinine will not ordinarily reduce Benedict’s solution,
under these conditions.
. Clinitest Tablet Tests
 Principle:
 - The same as that of Benedict's Qualitative Test.

 The clinitest tablet may be taught of as a solid form of

Benedict's Qualitative reagent.
 contains anhydrous sodium hydroxide, which results
in moderate boiling when added to dilute urine gives
heat in its reaction with citric acid
- Results are graded as negative, trace, 1+, 2+, 3+, or
4+ by comparison on color chart.
Clinitest Tablet Tests have;

 Two drop method

 Five drop method

 Sensitivity
Clinitest reagent tablets will detect as little as 250mg
of sugar in 100ml of urine.
Procedure for Five drop method
 Follow the direction supplied with the clintest tablets
 Place 5 drops of urine in a test tube and add 10 drops of distilled
water
 Add one clintest tablet by easing it in to the tube with out touching it.
It contains strong alkali.
 Watch while boiling takes place, but don’t shake or touch the bottom
of tube
 Wait 15 seconds after boiling stops, then shake the tube gently and
compare the color of the solution with the color scale
 Grade the results as negative, trace, 1+, 2+, 3+, or 4+. The results
correspond to the following concentrations (mg/dl): trace, 250mg/dl;
1+, 500mg/dl; 2+, 750mg/dl: 3+, 1000mg/dl: 4+, 2000mg/dl.
Specific (enzymatic) test for glucose

 These tests are based on the use of glucose oxidase

 The glucose oxidase in the presence of atmosphoric
oxygen oxidize glucose to gluconic acid and hydrogen
peroxide.
 The principle of all enzymatic test is the same
 They differ only on the uses of different type of
chromogen (a color indicative).
Specific (enzymatic) test for glucose

 Specific test for urinary glucose that will be discussed

here are :
 Clinistix reagent strip

 Tes tape

 Diastistix

 Which are available in the reagent strip

 Separately or in combination with other urine test
Principle of Clinistix

 It is a double sequential enzyme reactions

 Glucose oxidase will oxidize glucose to gluconic acid
and at the same time reduce atmospheric oxygen to
hydrogen peroxide.
 The hydrogen peroxide formed will, in the presence of
the enzyme peroxidase, oxidize the reduced form a
dye to the oxidized form, which is indicated by the
color change of an oxidation – reduction indicator
Clinistix Reagent Strip Test

Glucose + O2 Glucose oxidase Gluconic acid + H2O2

(in urine) (from air)

Step 2
H2O2 + reduced form of dye Peroxidase Oxidized form of dye + H 2O

(o- tolidine) (red) (oxidized o- tolidine) (blue

Chemical Reaction Chart
Chemical Exam

 When the test strip is

dipped in urine the
reagents are activated
and a chemical
reaction occurs.
 The chemical reaction
results in a specific
color change.
Positive Glucose

(Normal for comparison)

Procedure

Follow the directions supplied with the strips.

1. Rapidly dip the test end of the strip in the urine.
2. Read the results after exactly 10 seconds
3. Record the results as positive or negative.
If the test area remains red, the result is negative.
A positive result is indicated by the appearance of a purple
color in the test area.
Test-Tape for Glucose

 Principle
 Tes-Tape is a screening test
 specific for glucose.
 The principle is the same to clinistix
 Differ in the oxidation -reduction indicator employed, and
the material the reagents are impregnated on.
 In Tes-Tape the reagents are impregnated on a tear strip
of special paper, and the indicator is yellow in its
reduced form and green to blue in its oxidized form.
Procedure
 Follow the manufacture direction:
1. Tear off approximately 1 and 1/2 inch.
2. Dip part of the tape into the urine specimen; remove it
immediately.
3. Wait for 30 seconds; then observe the appearance of any
green color.
4. Record the result as positive or negative.

 If the test area remains yellow after 30 seconds, the

result is negative.
 If any green color is present at this time, the result is
positive.
Diastix Reagent Strip for Glucose

 Diastix is a specific test for glucose based on the use of

glucose oxidase,
 The chemical reaction is the same as clinistix
 the difference being the chromogen system used to
indicate the presence of glucose.
 The reagent area contains glucose oxidase, peroxidase,
a blue background dye, and potassium iodide as the
chromogen.
Procedure

 Follow the directions supplied with the reagent strip.

1. Dip the reagent area of the strip briefly into the
specimen.
2. Compare the test area with the color chart after 10
seconds to see whether the reaction is positive or
negative for glucose.
3. Compare the test area with the color chart at 30
seconds, for a semi-quantitative result, and report the
results as indicated on the chart.
 Rubner’s Test

 To 10 ml of undiluted urine add 3 gms (excess) lead

acetate, Shake well, Filter
 Boil the filtrate and add 1 ml concentrated ammonium
hydroxide and boil. The precipitate formed is the
criterion of the test.
 Lactose: Red solution + Red precipitate
 Glucose: Red solution + Yellow precipitate
 Fructose

 Fructose may be found in the urine mostly after fruits,

honey, syrup and jams food.
 It may be found in liver disease and along with glucose,
in the urine of diabetics.
Seliwanoff’s Test for Fructose

 Hydrochloric acid acts on fructose to form a derivative of

furfuraldehyde, which gives a red-colored compound
when linked with resorcinol.
 Seliwanoff’s Reagent
Dissolve 50 mg resorcinol in 33 ml concentrated
hydrochloric acid and dilute to 100 ml with water.
Procedure for Seliwanoff’s Test
 
 Add a few drops of urine (about 0.5 ml) to about 5 ml of
the reagent in a test tube
 Boil.
 Interpretation

Fructose gives a red color within half a minute. The test is

sensitive to 5.5 mmole/l (100 mg/100 ml) if glucose is
absent, but if glucose in present it is less sensitive and in
addition large amount of glucose can give a similar color.
Clinical Significance

 Normally, urine doesn't contain a sufficient amount of

sugar to react with any of the popular enzyme or
reducing tests.
 When sugar appears in the urine, it shows the
abnormality caused by disease diabetes mellitus.
 Test for urine sugar is used in screening to detect
diabetic mellitus, confirming diagnosis of D.mellitus, and
monitoring the effectiveness of diabetic therapy.
 Ketones
 Ketone bodies, also called Ketones,
 are a group of three related substances

 Acetone
 acetoacetate (acetoacetic acid) and
  -hydroxybutyrate ( -hydroxybutyric acid).
 Formed in liver from aceto acitate (oxidation product of
amino acid,faty acid)
 Used as energy source for extra hepatic tissue like brain
Ketone bodies an alternative fuel for cells.

 Liver mitochondria have the capacity to divert any

excess acetyl CoA derived from fatty acid or pyruvate
oxidation into ketone bodies.
 The compounds categorized as ketone bodies are
acetoacetate, 3- hydroxybutyrate and acetone (a volatile
non-metabolizable side product).
 They are transported in the blood to peripheral tissues,
where they can be reconverted to acetyl CoA and
oxidized by the TCA cycle.
cont’d

 Ketone bodies are soluble in aqueous solution, and

therefore, do not need to be incorporated in lipoproteins
or carried by albumin as do other lipids.
 They are produced in the liver during periods when the
amount of acetyl CoA present exceeds the oxidative
capacity of the liver.
cont’d

 The liver actively actively produces ketone bodies, but it

cannot itself use them as fuels.
 Ketone bodies are used in extrahepatic tissues such as the
skeletal and cardiac muscle and the renal cortex, in proportion
to their concentration in blood. Even the brain can utilize
ketone bodies for fuel if the level rises sufficiently, this is
important during prolonged periods of fasting.Extraheptic
tissues efficiently oxidize acetoacetate and 3-hydroxybutyrate.
cont’d

 The liver lacks succinyl CoA : acetoacetate CoA transferase

( thiophorase ), and therefore is unable to use acetoacetate as
a fuel.!!
 Red blood cells cannot efficiently oxidize acetoacetate and 3-
hydroxybutyrate because they lack mitochondria
Tests for ketone bodies in Urine

 Rothera’s test
 Lang’s test
 Acetest tablet test
 Acetone powder test
 Reagent strip tests (eg. ketostix)
Rothera’s test

Principle
 Both acetone and acetoacetic acid give a purple color
with alkaline sodium nitroprusside.
 This is the general principle for the tests mentioned
above.
  Results - Report the test as positive or negative
Procedure

1. To 5 ml of fresh urine, add ammonium sulphate crystals

until saturated (about 1 g.).
2. Add 2 drops of sodium nitroprusside reagent and mix .
3. Overlay with ammonium hydroxide solution (28%).
4. If acetone or acetoacetate is present, a red to purple
color will develop at the line of contact.
The color may not appear for 10-15 minutes.
5. Report the test as positive or negative.
Ketostix Reagent Strip Test

 Principle
 Is based on Rothera’s test,
 Acetoacetic acid will react with sodium nitroprusside in
a alkaline medium to form a purple color.
 If glycin is added, the test is slightly sensitive to acetone.

N.B Multistix and ketostix hav been formulated to react only

with aceto acetic acid. They don’t react with acetone.
Ketostix Reagent Strip Test

 Procedure
 Dip test- end of the strip in urine
 At 15 seconds compare the color of dipped- end with
the color chart.
 Report as indicated by the color chart.

 Content of Reagent Strip

 Sodium Nitroprusside, Glycine, and a strongly
Alkaline buffer.
Positive urine Ketones

(Normal for comparison)

Gerhardt’s Test for acetoacetate
 It is specific for acetoacetate ;
 however it is capable of detecting only large amounts of
acetoacetate.
 The test has been used as a means of determining the
severity of ketosis.
 A positive result indicates severe ketosis,
 and treatment must be started immediately.
Gerhardt’s Test for acetoacetate

Principle of the Test

 When acetoacetate reacts with a ferric chloride (FeC13)
solution, Bordeaux red color is formed.

 Reagent
 10% Ferric chloride reagent
 Weigh 10 g of ferric chloride and transfer to a 100 ml
volumetric flask.
Clinical Significance
When the rate of formation of ketone bodies is greater
than the rate of their use, their levels begin to rise in
the blood, which is called ketonemia, and eventually
in the urine, which is known as ketonuria.
These two conditions are seen most often in cases of
starvation and diabetes mellitus.
Ketone bodies can be seen also in the urine during
prolonged vomiting, severe diarrhea, anesthesia,
severe liver damage, high fat intake and low
carbohydrate diet.
Clinical Significance continued

 The excessive production and accumulation of ketone

bodies may lead to ketosis.
 Its physiological effect is serious because acetoacetic
acid and -hydroxybutyric acid contribute excess
hydrogen ions to the blood, resulting in acidosis - a
condition that tends to lower the blood pH. If not
corrected in time this may result in death.
 Another physiological effect found to be toxic to brain
tissue when present in increased amounts in the blood.
So this condition can result in permanent brain damage.
Determination of Urinary Protein
Introduction:
 Test for urinary protein is one of the most important and
valuable parts of the routine urinalysis.
 Albumin is one of the important proteins, which appears
in urine during a pathological condition.
 It often occurs as a symptom of renal disease.
 Globulins are excreted less frequently.
 Bence Jones protein is a specific type of globulin
excreted in multiple myeloma.
Causes of Proteinuria
1. Increased permeability of the glomerulus
 Normally, the glomerular membrane,in the formation
of urine, is not permeable for protein molecules.
 If the glomerular membrane is damaged these large
protein molecules can pass through
2. A decrease in normal reabsorption in the tubules
disease condition which affect reabsorption
Types of proteinurea

 classified according to the relationship of its etiology to

the kidney and also the mechanism involved.
 Functional Proteinuria
 Systemic disease or renal pathology
Proteinuria
A. Functional Proteinuria: not associated with renal
damage.
1. Accidental or false proteinurea:
 occurs when mixing of urine with a proteinous
fluid such as pus, blood or vaginal discharge
Functional Proteinuria cont’d…

2. Physiological proteinuria:
 It is usually less than 0.5 gm/24 hr. which is
associated with fever, exposure to heat or cold,
emotional stress, and later stage of pregnancy.
3. Posatural (orthostatic) proteinurea:
 associated only with the upright position or sitting
for a long time. The protein urea is intermittent and
disappears when the individual lies down
B. Systemic disease or renal pathology
Proteinuria.

1. Pre renal protein urea: not due to primary renal disease

 Fever and a variety of toxic condition
 Venous congestion
 Renal hypoxia
 Hypertension
 Myxedema
 Bence Jones protein
Systemic disease or renal pathology
Proteinuria cont’d…
2.Renal proteinuria: primarily kidney disease
 Gomerulonephritis
 Nephrotic syndrom, primary and secondary
 Destructive parenchymal lesions (tumour,
infection
Systemic disease or renal pathology
Proteinuria cont’d…

3. Post renal protein urea:

 protein added to the urine at some point farther down the
urinary tract from the renalparenchyma..
 Infection of the renal pelvis or urete
 Cystitis
 Urethritis or prostatitis
 Contamination with vaginal secretion
Test for urinary protein

1. Precipitation or turbidimetric test

 Principle: protein is either precipitated out
of the urine specimen by means of a chemical,
or it is coagulated out of solution with heat.
These tests include:
 Robert’s Test
 Heller’s Test ring tests
 Sulphosalicylic Acid Test non-ring tests
 Heat and Acetic Acid Test
Robert's Test

 Principle
The principle of this test is based on the precipitation
of protein and formation of white compact ring
using concentrated Nitric acid (HNO3).
Procedure of Robert's Test

 Place 3-5 ml urine in a test tube.

 Place the Robert's Reagent to the bottom of the tube and
allow 3 ml of the reagent to lay beneath the urine.
 A white ring at the zone of contact indicates a positive
test.
 The ring must be read within 3 minutes
 Report the result according to the type of the ring formed
Heller's Test

 Principle : The same as Robert`s Test

 Heller's Reagent: It is concentrated nitric acid.

 Procedure the same as roberts test

Sulphosalicylic Acid Test

 Principle
 Sulphosalicylic acid solution (20%) precipitates any
protein in the urine specimen.
 It is an anion precipitant that works by the
neutralization of the protein cation.
 This method is more sensitive and reliable than the
heat method.
 It can detect 5 to 10 mg/dl of protein in urine
specimen
Procedure of Sulphosalicylic Acid Test

 Take 2 ml of a centrifuge (clear) urine specimen in a

test tube.
 Add equal amount of Sulphosalicylic acid reagent.
 Shake the test tube gently and let stand for 10 minutes
 Note the degree of turbidity by looking at the
illuminated tube against a dark background
 Grade and report the result as non ring test
 interpretation: report the result based on the turbidity
formed
 Heat and Acetic Acid Test

 Principle: The test is based on the precipitation of protein

by heat.

 Procedure
 Fill a test tube three-fourth full of clear urine, and gently
heat the upper portion of urine for 2 minutes to boil,
being careful not to shake the tube more than
necessary. The lower portion of urine is not heated
so that it can be used as a control for
comparing.
 If turbidity ( a white cloud ) can arise due either of
phosphates, carbonates, or protein.
Procedure Cont’d…

 Add 3 drops of 10% acetic acid drop by drop, boiling

between each drop.
 A white cloud that disappeared is due to phosphates
or carbonates. Persistence or development of
turbidity implies proteinuria.
 Read the test and record results according to the
chart for non-ring precipition test.
Heat and Acetic Acid Test cont’d…

 Sensitivity
This method is the most sensitive for small amount of
protein and can reliably detect protein concentrations of
2 to 3 mg/dl.
Reagent Strip Reactions

 uses the principle of the protein error of indicators to

produce a visible colorimetric reaction.

 Contrary to the general belief that indicators produce

specific colors in response to particular pH levels, certain
indicators change color in the presence of protein even
though the pH of the medium remains constant. This is
because protein (primarily albumin) accepts hydrogen
ions from the indicator.
Reagent Strip Reactions

 The test is more sensitive to albumin because albumin

contains more amino groups to accept the hydrogen ions
than other proteins.
 Depending on the manufacturer, the protein area of the
strip contains either tetrabromphenol blue or 3′, 3′′, 5′, 5′
′-tetrachlorophenol-3, 4, 5, 6-tetrabromosulfonphthalein
and an acid buffer to maintain the pH at a constant level.
 At a pH level of 3, both indicators appear yellow in the
absence of protein
Colorimetric Reagent Strip (Dipstick)

 Principle
 The Colorimetric reagent strip test is based on the
ability of protein to alter the color of some cid-
base indicators without altering the pH.
 When an indicator, such as tetrabromophenol blue
is buffered at pH 3, it is yellow in solutions without
protein.
 Tests are more specific.
 They require only a drop of urine enough to
moisten the reagent area.
Procedure of Reagent Strip

 Observe the precautions and follow the

instructions supplied by the manufacturer.
1. Dip the reagent area of the strip briefly into the
specimen.
2. Remove excess urine by tapping or drawing the edge
of the strip along the rim of the urine container.
3. Compare the color that develops with the color chart
supplied by the manufacturer and report as indicated
on the chart.
Chemical Exam

 When the test strip is

dipped in urine the
reagents are activated
and a chemical
reaction occurs.
 The chemical reaction
results in a specific
color change.
Quantitative 24 hour protein Determinations

 estimates the protein content of urine.

 performed by quantitating the amount of precipitation
formed following the addition of a specific chemical to
the urine.
 The precipitate is measured either by comparison with
known standards (sulphosalicylic acid turbidity test) or
 By recording the height of the column of precipitate in a
specially-designed tube (Esbach's test).
Quantitative 24 hour protein Determinations

A. Sulphosalicylic Acid Turbidity Test

1. Pipette 2.5 ml of urine into a test tube.
2. Add 7.5 ml of 3% sulphosalicylic acid.
3. Invert to mix
4. Let stand 30 minutes.
 Compare the turbidity with known standards
prepared from solutions containing 10, 20, 30, 40,
75 and 100mg albumin/dl, and estimate the
concentration of the unknown (urine protein).
 If the unknown urine contains more than 100mg/dl
protein, dilute the urine and repeat the test.
Quantitative 24 hour protein Determinations

B. Esbach’s Test
1. measure the total volume; then filter some of the
urine.
2. Do qualitative protein test, Robert’s or strip test.
 if the urine is +3, made 1:5 dilution.

 if the urine is +4 make 1:10 dilution.

 if the urine is trace, +1 or +2 non dilution is

needed.
 if the urine is negative, a quantitative test is not
done.
 N.B: measure the pH of the urine. It should be acidic.
If not, add 10 % acetic acid
Esbach’s Test Cont’d…

4. Add pumice powder to the 0.5 mark of the Esbach’s

tube.
5. Add urine to the “U” mark.
6. Add Esbach’s reagent to the “R” mark.
7. Mix slowly by inversion, 10 times.
8. Wait for 30 minutes. Read the highest of the column.
Do not subtract the amount of the pumice.
9. The result is now in gram per liter of protein in the
urine. If the urine has been diluted, multiply by the
dilution factor, calculate, and record the g % and the g /
24 hrs.
Clinical Significance

 Proteinuria: >6-8gm/l.
 renal damage cause proteinuria.
 A positive protein test for urine may b correlated with
findings of casts.
 In cases of renal disease, it is essential that the
diagnosis be made and treatment started as soon as
possible to prevent extensive and permanent renal
damage.
 Proteinuria with the presence of leukocytes and bacteria
in the urine will be suggestive of urinary tract infection.
Bence - Jones Protein

 It is an abnormal low molecular weight globulin

consisting of light chains of immunoglobulin, which
contains the amino acid found in other proteins except
methionine.
 It may be found in the urine of patients with multiple
myeloma, which is a malignant disease of the plasma
cells, mainly affecting bone.
 The incidence of Bence-Jones proteinuria in multiple
myeloma has been estimated as 50% to 80%.
Heat test to screen Bence -Jones Protein

 Principle:
Bence Jones protein coagulates when heated to 40 to
600C and re-dissolves partially or wholly on boiling.
Albumin coagulates above 600C and does not re-
dissolve on boiling.
 Heat test to screen Bence -Jones Protein
Procedure
1. Take 5ml of clear fresh urine into each of three test tubes.
2. Acidify the urine by adding 1 drop of glacial acetic acid to
the first tube, 2 drops to the second tube, and 3 drops to the
third tube.
3. Place the three tubes in a beaker of tap water and insert a
0-100 OC thermometer in one of the tube.
4. Using a gentle flame slowly heat the beaker of water.
5. When the temperature reaches 40OC look for cloudiness
and precipitate in the tubes. Continue to observe until the
temperature reaches 60OC.
Clinical significance

 Suggests the diagnosis of multiple myeloma, leukemia or

waldenstorms macroglobulinemia.
 Interfering factor:
 False positive result may occur in connective tissue
disease, renal insufficiency, contamination of the
specimen with menstrual blood and prostatic
secretion or semen
 coagulable protein denatures or decompose at room
temperature
 Determination of Hemoglobin

 Hemoglobin is pigment in red blood cells composed of

an iron- containing group (hem) and a complex protein
(globin).
 it serves as a transporter of oxygen in the blood from
the lung to metabolically active tissues
 Hemoglobin appears in the urine when there is extensive
or rapid destruction (hemolysis) of circulating
erythrocytes that the reticuloendothelial system cannot
metabolize or store the excessive amounts of free
hemoglobin.
 Normal range 1-1.4g/l
Clinical significance

 Hemoglobinuria may occur with severe intravascular

hemolysis
 when the amount of hemoglobin being released into the
plasma is more than can be taken up by haptoglobin (the
plasma protein that binds free hemoglobin to prevent it
being lost from the body).
 This results from a variety of conditions and disease
states.
 It may be the result of hemolysis in the blood stream, in
a particular organ, in the kidney of lower urinary tract or
in the sample itself.
Causes of Intravascular Hemolysis

 Intravascular Hemolysis is associated with:

 Hemolytic anemia.
 Severe infectious disease such as Falciparum
Malaria, Yellow Fever, Small Pox and Typhoid
Fever.
 Glucose - 6- phosphate dehydrogenase (G6PD)
deficiency.
 The ingestion of certain drugs.
 Escherichia coli septicaemic.
Causes of Intravascular Hemolysis Cont’d…

 Incompatible blood transfusion.

 Snake bites that cause acute hemolysis.
 Sickle cell disease- crisis with sever hemolysis.
 Severe viral hemorrhagic fever accompanied by
intravascular hemolysis.
 Poisonings with strong acids or mushrooms sever-
burns and renal infarction.
Determination of Hemoglobin

 Commonly used Tests for the detection of hemoglobin in

urine :
 Benzidine
 Guaic
 Occultest
 Reagent strip test
Determination of Hemoglobin cont’d…
Principle
 Presence of peroxidase activity of the hemoglobin and
hydrogen
peroxide (H2O2) which librates oxygen.
 The intensity of the color depends on amount of librated
oxygen.
 The amount of librated oxygen depends on the peroxidase
activity of hemoglobin molecule, which intern depends on
the amount of hemoglobin found in the urine.
Principle cont’d…

 reactions are summarized :

 Hemoglobin + H2O2  Oxygen
 Oxygen + Chromogen  Blue or green oxidation
Factors that Affect Hemoglobin
Determination
 False negative
 High specific gravity such as heavy Proteinuria (over
5 g/1). This prevent lysis of RBCs and may reduce
the color reaction.
 Low to false negative results are obtained if the urine
contains large amounts of ascorbic acid.
 Nitrite delays test reaction.
 Formaline used as preservative, fix the cell and
prevent hemolysis.
Cont…

 False positive
 Low specific gravity < 1.010 enhances lysis and
produces color reaction.
 Microbial peroxidase produces false the color
reaction.
 presence of contaminating oxidizing detergents on
the urine such as bleach.
 Benzidine Test

 Principle
 The peroxidase activity of hemoglobin
decomposes hydrogen peroxide and the
liberated active oxygen oxidizes the organic
compound Benzidine.
 Procedure
 (1) Mix equal parts (1mL each) of A and B in a test tube
(15-mL) just before use.
(2) Add 2mL of urine (previously boiled and cooled to avoid
false
positive reaction).
 (3) The appearance a green or blue color with in 5minutes
indicates
the presence of  blood.
Report as :
Trace = faint green
1+ = green
2+ = greenish blue
3+ = blue
4+ = deep blue
 Occultest (tablet method)

 Principle
 When the tablet is moistened with water tartaric acid
and calcium acetate react with strontium peroxide to
form hydrogen peroxide.
 The hemoglobin in the urine catalytically decomposes
hydrogen peroxide liberating oxygen, which oxidizes
orthotolidine to a blue derivative.
Procedure

1.Place a piece of filter paper, which comes with the

reagent on a clean surface.
2.Place one drop of well-mixed urine in the middle of filter
paper.
3. Place Occultest tablet in middle of the moist area.
4. Flow two drops of water over the tablet.
5. Observe color of filter paper around tablet exactly two
minutes later.
6. The test is positive, if a blue color appears on the filter
paper around the tablet.
7. Report as positive or negative.
Reagent Strip Tests (Hemastix)

 Principle:
 The hemoglobin test spot of the reagent strip contains a
buffered mixture of organic peroxide and a chromogen
(o-toluidine, this is different from o-tolidine).
 The color of the chromogen changes in presence of
hemoglobin.
 Presence of high concentration of ascorbic acid or
vitamin C in a patient’s urine may inhiabit or delay the
color reaction for hemoglobin as the ascorbic acid acts as
the oxygen acceptor instead of the chromogen
Procedure

1. Dip the test end of strip into a well-mixed specimen of

urine and remove            immediately.
2. After 30 seconds compare the test area with the color
chart provided.
3. Report as indicated on the color chart.
Interpretation

 The color of the test spot for hemoglobin turns blue in

case of Hemoglobinuria, which is often associated with
hematuria. The latter is recognized by the presence of
red blood cell in the urinary sediment.
 The test strip cannot differentiate between
hemoglobinuria and haematuria but its positive reaction
(specklet pattern) alarms the technician to look for
haematuria, which is a sensitive, early indicator of renal
disease.
Clinical significance

 hemoglobinuria :The presence of free Hemoglobin in the

urine
 hematuria- a condition when intact red blood cells are
present in the urine.
 Hematuria is used to indicate bleeding somewhere in
the urinary tract.
 Usually both red blood cells and hemoglobin mark this
disorder
Cont…

 Hemoglobinuria may occur with severe intravascular

hemolysis
 Specimen
 Urine containing hemoglobin appears brown or
brown-gray on color and is usually cloudy.
 hematuria can be distinguished from hemoglobinuria
by a microscopic examination
 It should be performed as soon as possible
 Urine Myoglobin

 Myoglobin - a red pigment found in the cytoplasm of

cardiac and skeletal muscle cells in the urine.
 Myoglobinuria occurs when muscle cells are extensively
damaged, as by disease or sever crushing trauma .
Myoglobin Cont’D…

 Principle: urine mixed with ammonium sulphatea and it

becomes dissolved and measured.
 Interfering factor
 Ingestion of large amount of vitamin C
 Extremely dilute urine can reduce test sensitivity
 Contamination of urine with iodine during surgery may
produce positive result
Clinical Significance

 To aid diagnosis of muscle disease

 To detect extensive infarction of muscle tissue
 To assess the extent of muscular damage from
crushing trauma
Determination of Bilirubin
Production of Bilirubin

 Bilirubinis a waste product that must be eliminated

from the body.
 On its way to the liver it is not water-soluble and is
carried through the blood stream linked to plasma
albumin as a bilirubin-albumin complex.
 This water-insoluble form of bilirubin is often referred
to as free bilirubin or unconjugated bilirubin
Production of Bilirubin

 Bilirubin is normally excreted from the body by the

liver through the intestine.
 It can not be excreted by the kidney because free
bilirubin linked to protein can not pass through the
glomerular capsule.
 In the liver, bilirubin is converted to a water-soluble
product by the kupffer cells of the liver.
 It is made soluble by conjugation with glucuronic acid
and other hydrophilic substances to form bilirubin
glucuronide
Production of Bilirubin

 The water-soluble form is conjugated (direct)

bilirubin.
 Being water soluble, conjugated bilirubin can be
eliminated from the body by way of the kidney or the
intestine.
 Once conjugated, bilirubin is normally excreted by the
liver into the bile, transported to the common bile duct
and then to the gallbladder, where it is concentrated
and emptied into the small intestine.
Production of Bilirubin

 Here bilirubin is converted to urobilinogen, which is

colorless, and eventually eliminated from the body in
feces as a colored oxidation product (stercobilin).
 of the urobilinogen formed, about half is normally
reabsorbed into the portal circulation and
subsequently excreted again by the liver into the
intestine.
 Therefore bilirubin is normally excreted from the body
by the liver and eliminated by the way of intestine.
Clinical Significance
 Conjugated bilirubin appears in the urine when the
normal degradation cycle is disrupted by obstruction of
the bile duct (e.g., gallstones or cancer) or when the
integrity of the liver is damaged, allowing leakage of
conjugated bilirubin into the circulation.
 Hepatitis and cirrhosis are common examples of
conditions that produce liver damage, resulting in
bilirubinuria.
 Jaundice due to increased destruction of red blood cells
does not produce bilirubinuria. This is because the
serum bilirubin is present in the unconjugated form and
the kidneys cannot excrete it.
Methods of determination of bilirubin

 Harrison’s (Faucet's) Test.

 Barium chloride filter paper method
 Diazotization Test for bilirubin
 Icto test Tablet test
 Harrison’s (Fouchet’s) Test

 Principle
 Barium chloride reacts with sulphate radical in the
urine to form barium sulphate.
 If bilirubin is present in the urine, it adheres to the
precipitate and is detected by the oxidation of bilirubin
(yellow-colored) to biliverdin (green-colored) on
treatment with ferric chloride (Fouchet’s reagent) in the
presence of trichloracetic acid.
 A blue color is given by bilicyanin.
 Harrison’s (Fouchet’s) Test Procedure

 Positive: Yellow
 Check the pH of the urine with a litmus paper strip.
 It should be slightly acidic.
 If alkaline, acidify with a few drops of 33% acetic acid.
 Take 10ml of the centrifuged urine and add to that 5ml
of 10% barium chloride.
 Mix well.
 It will become milky with white or yellow precipitate. If
the precipitate formed is insufficient, add a drop of dilute
sulphuric acid or ammonium sulphate solution.
 Filter the precipitate through a small size filter paper .
 Procedure Cont…
 take the paper out and carefully unfold it on a dry filter
paper to soak the fluid and place both on a white tile or
wax paper.
 Note the color of the precipitate and make the preliminary
observation:
 Negative: White or nearly white
 Add one drop of Fauchet’s reagent onto the precipitate in
the center of the filter paper holding the precipitate.
 If bile is present, a green or blue color develops; the
color intensity is proportional to the amount of bile
pigment present.
 Negative: No change in color
 Pale blue-green:Trace or 1+
 Darker blue-green: 2+ to 4+
 Barium chloride filter paper method

 It is the modification of Harrison’s test.

 The barium chloride is supplied on thick filter paper that
has been soaked in a saturated solution of barium
chloride.
 Principle
 The principle of the test is the same as Harrison’s
(Fouchet’s) Test
 Reagent
 Soak thick filter paper in a saturated barium chloride.
Dry and cut in to small strips (4x½ inch strips)
 Diazotization Test for bilirubin

 Principle:
 the tablet and reagent strip tests for bilirubin are
based on the coupling of bilirubin with a diazonium
salt (p-nitrobenzene diazonium p-toluen sulfonate) in
an acid medium to form azobilirubin, which gives a
blue or purple color.
Reagent Strip (Diazo) Reactions

 testing for urinary bilirubin by reagent strip uses the diazo reaction.
 Bilirubin combines with 2,4-dichloroaniline diazonium salt or 2,6-
dichlorobenzene-diazonium-tetrafluoroborate in an acid medium to
produce an azodye, with colors ranging from increasing degrees of
tan or pink to violet, respectively.
 Qualitative results are reported as negative, small, moderate, or
large, or as negative, 1, 2, or 3.
 Reagent strip color reactions for bilirubin are more difficult to
interpret and are easily influenced by other pigments present in the
urine.
 bilirubin glucuronide + diazonium salt → azodye
 Icto test Tablet test
 Principle
 Ictotest tablet test is based on a diazo reaction, in
which bilirubin is coupled with a diazonium salt in an
acid medium.
 The tablets are supplied with a special absorbent mat.
 The tablet contains the reactive ingredients.
 The tablet is place over the urine on the mat, and
water is added to the tablet.
 When bilirubin is present, it reacts with a solid
diazonium salt present in the tablet to form azobilirubin.
 A positive reaction is seen as a blue or purple color on
the mat.
.
 Procedure
 Place 10 drops of urine on the centerl test mat
 Place the Ictotest tablet in the center of the urine-moistened area.
 Place 1 drops of water onto the tablet. Wait 5 seconds; then place
a second drop of water onto the tablet so that the water runs off the
tablet onto the mat.
 Remove the tablet, and observe the mat for the appearance of a
blue to purple color at 60 seconds.
 Negative: The mat shows no blue or purple within 60 seconds.
 Positive: The mat around or under the tablet turns blue or
purples, within seconds.                       
 Sensitivity:Ictotest detects 0.1mg of bilirubin in 100ml of urine.
Determination of Urobilinogen

 Introduction
 Inthe intestine, most of the bilirubin is converted to
urobilinogen or stercobilinogen by the action of certain
bacteria that make up the intestinal flora.
 Approximately half of the urobilinogen formed in the
intestine is absorbed into the portal blood circulation
and returned to the liver.
 In the liver most of the urobilinogen is excreted into
the bile once again and returned to the intestine.
Urobilinogen int. cont’d…

 A very small amount of urobilinogen is excreted from

the body in the urine as urobilinogen or can be also
converted into urobilin, which gives the urine its
characteristic color with the other color pigments
(urochroms).
 Urobilinogen is also converted into urobilin when
exposed to air.
Urobilinogen int. cont…

 Stercobilinogen in the intestine is either eliminated

from the body unchanged or oxidized to the colored
compound stercobilin, which gives the faces its
characteristic color.
 Thus, urine normally contains only a very small
amount of urobilinogen and no bilirubin.
 Both are abnormal urinary constituents
Urobilinogen int. cont…

 When testing for urobilinogen the urine specimen

must be fresh, since it is usually unstable and it is
rapidly oxidized to urobilin.
 The presence of urobilinogen and that of urobilin have
the same clinical significance, however, they take part
in different chemical reactions, and urine is more
frequently tested for urobilinogen.
Test for urobilinogen

 EhrlichTest
 Wallace-Diamond quick screen method
 Reagent Strip Tests for urobilinogen-uroblistix
 Semiquantitation of urobilinogen
 Ehrlich Test

 Principle: The test depends upon the reacrtion

between urobilinogen and paradimethylamino
benzakdehide to form a chery (deep) red color.
 This is good for the screening test, but prophobilinogen,
like urobilinogen, also gives a red color with Ehrlich’s
reagent.
 This is differentiated by the addition of sodium acetate,
which alters the pH and intensifies the color given by
urobilinogen but not by porphobilinogen.
 Ehrlich Test cont’d…

 Procedure:
1. Place 10 ml urine in a test tube.
2. Allow warming to room temperature.
3. Add 1 ml Ehrlich's reagent and mix.
4. Let stand 3 to 5 minutes
 Normal amounts of urobilinogen present in the urine
sample will change the solution to pink.
 Abnormally high amounts of urobilinogen will change
the solution to a Cherry red color.
 This must be reported positive for urobilinogen
Ehrlich Test (cont…)

 Report as positive for urobilinogen

 Principle of Reagent Strip Tests:

 is based upon the reaction between p-
dimethylaminobenzaldehyde and urobilinogen to give
a deep-cherry red color.
Clinical Significance

 Increased urobilinogen may be seen in the urine in any

condition, with increased destruction red blood cells .
 It also may be seen in concentrated urine in fever and
dehydration.
 Urobilinogen in urine is associated with liver disease
and dysfunction.
 urobilinogen is found in the urine in conditions, such
infectious (viral) hepatitis, toxic hepatitis, cirrhosis,
congestive heart failure, and infections mononucleosis
with liver involvement.
Test For Urobilin

 Urobilin is an oxidation product of urobilinogen.

 Urobilin is colored and urobilinogen is colorless.
 Both compounds have the same clinical significance
when present in urine; however, they undergo
different chemical reactions.
 Schilesinger's Test for Urobilin

 Principle:
 When zinc acetate reacts with urobilin it produces a
green fluorescence.
 Reagent
 Alcoholic solution of zinc acetate for urobilin.
 Place 100ml of Ethyl in a beaker.
 Add zinc acetate with strong until no more goes in to
solution.
 Weigh 5gm of Iodine and 10gm of potassium Iodid.T
 transfer all reagents to a brown bottle.
 Porphobilinogen

 Introduction:
 Porphobilinogen is a normal, colorless precursor of the
porphyrin.
 Porphyrin are a group of compounds used in the
synthesis of hemoglobin.
 Porphyrins are normally eliminated from the body in the
urine and feces primarily as coprophyrin I with a small
amount of coproporphyin III.
 However, with certain inherited enzyme deficiencies
such as lack of porphobilinogen deaminase, there is
blockage in the normal pathway with increased
excretion of other porphyrins in the urine
 Hoesch Test for Porphobilinogen

 Principle:
 This is a test for porphobilinogen based on an inverse
Ehrlich’s aldehyde reaction.
 In the Hoesch test, an acid solution is maintained by
adding a small volume of urine to a relatively large
volume of Ehrlich reagents.
Hoesch Test for Porphobilinogen
cont’d…
 Reagent
1. Hydrochloric acid, 6mol/L.
 Dilute concentrated Hcl 1:2 with deionized or distilled
water by slowly adding the acid to water.
2. Hoesch reagent:
 Dissolve 2.0g P-dimethylaminobenzaldehyde in 6
mol/L Hcl & dilute  to 100ml with Hcl.
Clinical Significance

 Porphobilinogen is seen in the urine in acute attack of

acute intermittent porphyria, variegate porphria, and
hereditary coprophyia.
 An acute attack may be precipitated by drug affecting
the liver, such as certain anesthetics or barbiturates.
 The discovery of porphobilinogen in urine is a critical
value that can eliminate or reduce adverse effects
from drugs or anesthetics.
 Leukocyte Esterase

 Introduction:
 Measurement of leukocyte esterase is an indirect
measurement of leukocytes.
 Positive leukocyte esterase reactions in urine occur
most often with increased neutrophiles, which are
present in response to bacterial infections.
 The lymphocytes and epithelial cells do not certain
leukocyte esterase and are not measured in this test.
Leukocyte Esterase int. cont’d…

 Normal urine contains about 0 to 2 leukocytes per

high power field.
 The sensitivity of the reagent strip tests for leukocyte
esterase is about 5 to 15 leukocytes per high power
field.
 Therefore normal urine will give a negative reagent
strip reaction, yet the absence of leukocyte esterase
does not rule out a UTI.
 The results for leukocyte esterase are useful in
combination with tests for nitrite, pH and protein.
Reagent strip test for leukocyte esterase

 Principle
 Reagent strip tests utilize a diazo reaction. The test
area contains an ester that is hydrolyzed by leukocyte
esterase to form its alcohol (which contains an
aromatic ring) and acid.
 The aromatic ring is then coupled with a diazonium
salt, present in the test area, to form an azo dye, which
is seen as the formation of a purple color.

 Specificity:- the reaction is specific for esterase that is

present in granulocytic leukocytes. Primarily neutrophils
in urine
 Interferences
 False positive result
 Strong oxidizing agent like chlorine bleach and urinary
preservatives
 False negative or reduced result
 Drugs
 Elevated glucose (over 3 g/dl)
 High specific gravity
 Oxalic acid – metabolic of ascorbic acid
 High level of albumin (over 500mg/dl)
 Large amounts of ascorbic acid may inhibit the reaction
because of the reduction of the diazonium salts
 Clinical Significance
 When the urinary tract is infected at any point from the
urethra to the kidney, increased numbers of leukocytes,
especially neutrophils, are typical.
 These cells are also seen in the urinary sediment.
However, neutrophils are rapidly lysed or destroyed in
the urine, possibly a result of their phagocytic activity.
 Once lysed, they are undetectable in the microscopic
examination of the sediment.
 However, the reagent strip tests for leukocyte esterase
remains positive whether lysed or intact cells are in the
urine.
 Clinical Significance

 Increased leukocyte esterase also may be seen in the

absence of bacteria in the sediment or on routine urine
culture.
 Inflammatory conditions may result in increased
numbers of leukocytes without bacterial infection.
 Increased leukocytes may remain after treatment with
antibiotics, without bacteria.
 Immunosuppressed patients with UTI are unable to
produce adequate quantities of leukocytes in response
to their infection.
 Determination of Nitrate
 Reagent strip tests for nitrite are used as a rapid method
for detecting asymptomatic UTI.
 This test is most useful when combined with reagent
strip tests for leukocyte esterase.
 The detection of nitrite in the urine can be used to
indicate the presence of bacteria that reduce urinary
nitrate to nitrite.
 The presence of urinary nitrite indicates the existence of
a urinary tract infection.
Method Used for Detection of Nitrite

 Microscopic examination:
 urine sediment when examined microscopically can
reveal bacteria when present.

 Chemical dipstick
 Reagent Strip Test
 Principle
 Reagent strip test for nitrate are based on the Griess
test.
 This involves a diazo reaction. Nitrate will read with an
aromatic amino (P- arsanilic or sulfanilic acid) in an acid
medium to produce a diazonium salt.
 The diazonium salt is then coupled with another
aromatic ring (quinoline) to give an azo dye, which is
seen as a pink or red color.
Reagent Strip Test cont’d

Limitation:
 False positive reactions may be caused by bacterial
growth in ‘old’ urine specimen or by medication such
as Phenazopyridine that colors the urine red or that
turn red in an acidic medium.
Interference

 Fasle positive result

 In vitro conversion of nitrate to nitrite because of
bacterial contamination in an improperly collected or
stored urine specimen may give false positive result
 Medication such as phenazophyridine or other dyes
that color urine red or that turn red in an acidic
medium may mark color development or cause false
positive results
 Interference cont’d…

 False Negative results

 Insufficient incubation time in the bladder
 Insufficient dietary nitrate present in urine for bacteria
to reduce nitrate to nitrite
 Degradation of nitrite to nitrogen will give false
negative results
 Clinical Significance
 Most infections begins in the lower urinary tract as a
result of fecal contamination from the organism
normally present in the feces, such as E.coli
 The urinary tract is normally sterile.
 Infection is introduced via the urethra and ascends to
the bladder, ureters, and finally the kidney.
 Early detection and treatment is important to prevent
this ascending infection and subsequent renal
involvement.
 Detection of such infection is particularly important in
pregnant women and young girls to prevent
permanent renal damage.
Determination of Urinary Calcium
 Introduction:
 vitally important in maintaining the correct conditions for
normal neuromuscular transmission and glandular
secretion and for the activity of enzyme systems
particularly those involved in blood coagulation.
 The bulk of calcium ions (Ca++) discharged by body is
excreted in the stool. However, there is small quantity
of calcium that is normally excreted urine.
 But it may increase depending up on the quantity of
dietary calcium ingested.
Calcium Test (Sulkowitch Test)

 Principle
 In this test a solution of ammonium oxalate ( about
4 % ) is added to the urine. If calcium is present in
excessive amounts, it drops out of solution as a heavy
white precipitate of calcium oxalate.
 Procedure
1.Pour 5 ml of urine into a test tube
2. Add 5 ml of Sulkowitch reagent
3. Mix by inverting the tube several times
4. Allow to stand 3 minutes
5. If a fine white cloud appears, the calcium content is
normal
6. If no white cloud appears, the calcium content is
decreased
7. If a heavy white milky precipitate forms, the calcium
content is increased
8. Report the calcium content as normal, decreased or
increased
Interfering factors

 False positive
 High sodium and magnesium intake
 High milk intake
 If test done immediately after high calcium meal
 False negatives
 Increased dietary phosphates
 Alkaline urine
 Clinical Significance

 Hyperparathyroidism is a generalized disorder of

calcium, phosphate and bone that results from
increased secretion of parathyroid hormones and an
increased excretion of urinary calcium.

 Inhypoparathyrodism the urinary calcium is

decreased.
Determination of Indican

 Introduction:
 Indican is derived from indole, which is the
putrefaction product of protein, by protolytic bacteria.
 Patients with bacterial over growth in the small
intestine excrete large amount of metabolites of amino
acids such as tryptophan or tyrosine.
 Indole is produced by bacterial action on tryptophan
in the intestine, which mostly eliminated with feces,
some are absorbed and detoxified in the liver and
excreted as indican in the urine.
 Indican cont…
 It is increased in:
 High protein diet
 Pathological conditions such as
 Bacterial putrefactions.
 Enteritis.
 Pancreatic insufficiency.
 Intestinal Infection.
 Ulceration of intestinal mucosa.
Note: To differentiate the pathological conditions from non-
pathological, first restrict the patient from protein intake and
then do the test.
 Indican Tests ( Obemyares Test)

 Principle:
 HCL liberates indoxyl from indican and ferric chloride
(FeCl3) oxidizes the indoxyl to indigo blue.
 Procedure

1. Add 5 ml well mixed fresh urine and 5 ml obemyares

reagent into a test tube.
2. Add 4 drops of chloroform and mix several times by
inverting until all color dissolves out.
3. Add saturated ferric chloride drop by drop, mixing well
after each addition and record the number of drops you
add to decolorize
 Interpretation

 When indican is present, the chloroform layer shows a

deep violet to blue color.
 Normally when two drops are added, the color comes to
light sky blue, if it needs more, it shows the presence of
increased amount of indican.
Source of Errors

 Indican may be formed because of slow oxidation.

 Presence of iodide may case violet color removed by
adding crystals of sodium thiosulfate.
 Bile pigments – removed by shaking BaCl2 and
filtering
 Determination of Melanin
 Introduction:
 Melanin is pigment derived from tyrosine, which is normally present
in hair, skin and in the choroid layer of the eye
 Melanomas with pigments are normally transferred from
melanocytes to skin and mucus membrane cells.
 In patients with tumors arising from the melanin producing cells, the
melanomas, the melanin may be excreted in the urine in large
amount, and its presence is indicative of metastasis of the tumor to
the liver or other organ.
 The urine becomes black upon standing (oxidation), where the
chromogen /melanogen is changed into the pigment called melanin
which is a physical method to detect melanin.
 Chemical Tests for Melanin

 Three types of chemical test

a. Utilization of oxidizing agent (e.g. FeCl3)
b . Utilization of reducing action of melanogen
c. Oxidation by atmospheric air
Clinical Significance

 Melanin occurs in metabolic tambours especially

with metastasis of liver.
 Chemical Tests for Melanin cont’d
A. Ferric Chloride Test
Principle:
FeCl3 oxidizes the melanogen to melanin.
Procedure
1.To 5 ml of freshly voided urine, add 1 ml 10% FeCl3 drop
by drop to precipitate all the phosphates.
2. Add drop by drop 10% HCl to dissolve all the precipitate
and it forms different color.
3. Centrifuge and examine for black or gray precipitate of
melanin.
 Chemical Tests for Melanin cont’d
B. Thormahlel Test
 Principle
 Sodium Nitroprusside is reduced to ferocyanide
(Prussian blue) by reducing action of melanogen.
 Procedure

1. Prepare fresh solution of sodium nitroprusside by shaking

few crystals of sodium nitroprusside in 10 ml of d. water.
2. Add 0.2 ml aqueous sodium nitroprusside to 5ml urine
3. Add 0.5 ml of 40% sodium hydroxide. During this time the
urine turns red due to the action of creatinine and other
interfering substances.
4. Add 3.5ml of 30% glacial acetic acid, and the color may
change to blue or dark if melanin present which is
indication for a positive reaction.
 Chemical Tests for Melanin cont’d

C. Oxidation by Atmospheric Air

 Principle:
 Allow the urine sample to stand exposed to the air
undisturbed for 24 hours. If melanogen presents, it will
slightly oxidize to melanin by the air, and turns dark
brawn or black from the top downwards.
 Homogenestic acid gives the same effect, but the
darkness of melanogen is not accelerated appreciably by
alkali.
 Microscopic examination of sediment for melanin cast is
also possible
 ASCORBIC ACID

 Introduction:
 Ascorbic acid (vitamin C) is neither a normal nor a
pathologic constituent of urine.
 Ascorbic acid interfere with the various reagent strip
tests that are based on the diazo reaction.
 In these tests, the ascorbic acid may react with the
diazonium salt that is formed, causing a reduced or
false-negative reaction.
Reagent Strip Test for Ascorbic Acid

 Principle:
 The reaction is based on the reduction by ascorbic
acid of phosphomolybdate, a yellow complex, to
molybdenum blue.
 Specificity
 The reaction is not specific for ascorbic acid but will
react with other reducing substances with similar
redox potential
 Mucopolysaccharide (MPS)

 Introduction:
 Mucopolysaccharidosis in infants may lead to dwarfism
and mental retardation.
 In this genetic disorder mucopolysaccharide (a
conjugated protein with carbohydrate) is found in the
urine.
 Special test-strip is available for testing the presence of
mucopolysaccharide in urine.
 Exercises

1. Discuss by comparison the Benedict's Qualitative and

Glucose oxidase Tests.
2. List down the possible substances, which give false
positive results in non-specific tests for glucose
determination.
3. Mention the physiological effects of ketone accumulation
in blood.
4. Write the principle of the test for determination of bilirubin
and hemoglobin.
5. Write the general principles for the two types of
determination of urinary protein.
References:

 District laboratory practice in tropical countries. 2nd ed. Part I. Monica

Cheesbrough, 2005
 Text book of urinalysis and body fluids. Doris LR, Ann EN, 1983
 Urinalysis and body fluids: A color text and atlas. Karen MR, Jean JL.
1995
 Clinical chemistry: Principles, procedures, correlation. 3rd ed. Michael L.
Bishop et al. 1996
 Tietz Text book of clinical chemistry. 3rd ed. Carl AB, Edward RA, 1999
 Clinical chemistry: Theory, analysis, correlation 4th ed. Lawrence AK. 2003
 ASCP Document
 Urinalysis lecture note . Mistire W. , Dawite Y.

210
The next chapter will be on
Microscopic Examination Of Urine

What is microscopic of urine?

What is the importance of dealing
urine sediments?