URINE ANALYSIS

COMPOSITION OF NORMAL URINE

 INDICATIONS FOR URINALYSIS
1. Suspected renal diseases like glomerulonephritis, nephrotic
syndrome, pyelonephritis, and renal failure.

2. Detection of urinary tract infection.

3. Detection and management of metabolic disorders like diabetes

mellitus.

4. Differential diagnosis of jaundice.

5. Detection and management of plasma cell dyscrasias.

6. Diagnosis of pregnancy.
 COLLECTION OF URINE
 First morning, midstream: Preferred for routine urine
examination.
 Random, midstream: Routine urine examination.
 First morning, midstream, clean catch:
Bacteriological examination.
 Postprandial: Estimation of glucose, urobilinogen
 24-hour: Quantitative estimation of proteins or
hormones.
 Catheterised: Bacteriological examination in infants,
bedridden patients, and in obstruction of urinary tract.
 Plastic bag (e.g. colostomy bag) tied around genitals:
Infants, incontinent adults.
CHANGES WHICH OCCUR IN STANDING
URINE@ROOM TEMPERATURE
 Increase in pH due to production of ammonia from
urea by urease-producing bacteria.
 Formation of crystals due to precipitation of
phosphates and calcium (making the urine turbid)
 Loss of ketone bodies, since they are volatile.
 Decrease in glucose due to glycolysis and utilization
of glucose by cells and bacteria.
 Oxidation of bilirubin to biliverdin causing false-
negative test for bilirubin
 Oxidation of urobilinogen to urobilin causing false-
negative test for urobilinogen
 Bacterial proliferation
 Disintegration of cellular elements, especially in
alkaline and hypotonic urine.
Urine sample must be tested in the laboratory within 2 hours of
collection to get the correct results.
PRESERVATION OF URINE SAMPLE
 Refrigeration (4-6°C) is the best general method of
preservation of urine sample up to 8 hours.
 Following chemical preservatives can be added to the
24-hour urine sample:
 Hydrochloric acid: It is used for preservation of a 24- hour
urine sample for adrenaline, noradrenaline, vanillylmandelic
acid, and steroids.
 Toluene: It forms a thin layer over the surface and acts as a
physical barrier for bacteria and air. It is used for
measurement of chemicals.
 Boric acid: A general preservative.
 Thymol: It inhibits bacteria and fungi.
 Formalin: It is an excellent chemical for preservation of
formed elements.
 URINE EXAMINATION

 PHYSICAL EXAMINATION

 CHEMICAL EXAMINATION

 MICROSCOPIC EXAMINATION
 PHYSICAL EXAMINATION
Volume
 Average 24-hr urinary output in adults is 600-2000 ml.
 The volume varies according to fluid intake, diet, and climate.
 Abnormalities of urinary volume are as follows:
 Polyuria means urinary volume > 2000 ml/24 hours.
This is seen in diabetes mellitus, diabetes insipidus,
chronic renal failure, diuretic therapy.
 Oliguria means urinary volume < 400 ml/24 hours.
This is seen in febrile states, acute glomerulonephritis, congestive
cardiac failure or dehydration.
 Anuria means urinary output < 100 ml/24 hours or complete
cessation of urine output.
This occurs in acute tubular necrosis (e.g. in shock, hemolytic
transfusion reaction), acute glomerulonephritis and complete urinary
tract obstruction.
 PHYSICAL EXAMINATION
Color
 Normal urine color in a fresh state is pale yellow or amber and is
due to the presence of various pigments collectively called
urochrome.
 PHYSICAL EXAMINATION
Appearance

 Normal, freshly voided urine is clear in appearance.

 Foamy urine occurs in the presence of excess proteins
or bilirubin.
 Causes of cloudy or turbid urine
 PHYSICAL EXAMINATION
Odor

 Freshly voided urine has a typical aromatic odor due to

volatile organic acids.
 After standing, urine develops ammoniacal odor 
formation of ammonia occurs when urea is decomposed by
bacteria).
 Some abnormal odors with associated conditions are:
 Fruity: Ketoacidosis, starvation
 Mousy or musty: Phenylketonuria
 Fishy: Urinary tract infection with Proteus, tyrosinaemia.
 Ammoniacal: Urinary tract infection with Escherichia coli,
old standing urine.
 Foul: Urinary tract infection
 Sulfurous: Cystinuria
 PHYSICAL EXAMINATION
Specific Gravity (SG)

 Measure of concentrating ability of kidneys and is

determined to get information about this tubular function.
 It is basically a comparison of density of urine against the
density of distilled water at a particular temperature.
 Normal SG of urine is 1.016 to 1.022 in 24 hrs
sample and also depends on the state of hydration.
 SG of normal urine is mainly related to urea and
sodium.
 SG increases as solute concentration increases and
decreases when temperature rises (since volume
expands with rise in temperature).
 PHYSICAL EXAMINATION
Specific Gravity (SG)

 Hypersthenuria - Causes of increase in SG of urine

are diabetes mellitus, glycosuria, albuminuria, fever
and dehydration.
 Hyposthenuria - Causes of decrease in SG of urine
are diabetes insipidus, pyelonephritis, diuretics and
alcohol
 Isosthenuria - Low and fixed SG at 1.010 due to loss
of concentrating ability of tubules seen in end stage
renal failure.
PHYSICAL EXAMINATION
Specific Gravity (SG)

Urinometer
 PHYSICAL EXAMINATION
Specific Gravity (SG)
Urinometer
1. Fill a measuring cylinder with 50 ml
of urine.
2. Lower urinometer gently into the
urine and let it float freely.
3. Let urinometer settle; it should not
touch the sides or bottom of the
cylinder.
4. Take the reading of SG on the scale
(lowest point of meniscus) at the
surface of the urine.
5. Take out the urinometer and
immediately note the temperature of
urine with a thermometer.
6. For every 3°C increase / decrease
add / subtract 0.001
 PHYSICAL EXAMINATION
Specific Gravity (SG)
Refractometer method:

 SG can be precisely determined

by a refractometer, which
measures the refractive index of
the total soluble solids.
 Higher the concentration of
total dissolved solids, higher is
the refractive index.
 Extent of refraction of a beam • The method is simple and requires
of light passed through urine is only 1-2 drops of urine.
a measure of solute • Result is read from a scale or from
concentration, and thus of SG. digital display.
 PHYSICAL EXAMINATION
Specific Gravity (SG)
Reagent strip method:

 Measures the concentration

of ions in urine, which
correlates with SG.
 Depending on the ionic
strength of urine, a
polyelectrolyte will ionize in
proportion.
 This causes a change in
color of ph indicator
(bromothymol blue).
 PHYSICAL EXAMINATION
Reaction and pH
 pH is the scale for measuring acidity or alkalinity.
 Fresh urine is required for correct estimation of pH  upon
standing, urine becomes alkaline owing to loss of CO2 &
production of ammonia from urea.
 Normal value ranges from 4.6-8
 Urine pH depends on diet, acid base balance, water balance, and
renal tubular function
 Various methods for determination of reaction of urine:
 Litmus paper
 pH indicator paper
 pH meter
 Reagent strip tests
 PHYSICAL EXAMINATION
Reaction and pH
1. Litmus paper test:
 A small strip of litmus paper is dipped in urine and any color
change is noted.

2. pH indicator paper:
 Reagent area (impregnated with bromothymol blue and methyl
red) of indicator paper strip is dipped in urine sample and the
color change is compared with the color guide provided.
 Approximate pH is obtained.
 PHYSICAL EXAMINATION
Reaction and pH
3. pH meter:
 An electrode of pH meter is dipped in urine sample and pH
is read off directly from the digital display.
 It is used if exact pH is required.

4. Reagent strip test:

 The test area contains polyionic polymer bound to H+
 On reaction with cations in urine, H+ is released causing
change in color of the pH-sensitive dye.
 PHYSICAL EXAMINATION
Reaction and pH
Acidic urine Alkaline urine

 Ketosis  Severe vomiting

 diabetes mellitus  Old ammoniacal urine
 Starvation sample
 fever  Chronic renal failure
 Urinary tract infection by  Urinary tract infection by
Escherichia coli bacteria that split urea to
 High protein diet. ammonia - proteus or
pseudomonas
 Vegetarian diet
 URINE EXAMINATION

 PHYSICAL EXAMINATION

 CHEMICAL EXAMINATION

 MICROSCOPIC EXAMINATION
 CHEMICAL EXAMINATION

The chemical examination is carried out for the following

substances :-
 Proteins
 Glucose
 Ketones
 Bilirubin
 Bile salts
 Urobilinogen
 Blood
 Hemoglobin
 Myoglobin
 Nitrite or leukocyte esterase
 CHEMICAL EXAMINATION
Proteins
 Normally, kidneys excrete scant amount of protein in
urine
(up to 150 mg/24 hours)
 These proteins include
 proteins from plasma (albumin)
 proteins derived from urinary tract
 Tamm-Horsfall protein*
 secretory IgA
 proteins from tubular epithelial cells, leucocytes and other
desquamated cells.
 This amount of proteinuria cannot be detected by
routine tests.
*normal mucoprotein secreted by ascending limb of the loop of Henle.
 CHEMICAL EXAMINATION
Proteins
 Proteinuria refers to protein excretion in urine >150
mg/24 hours in adults.
 Causes
 Glomerular proteinuria due to increased permeability
of glomerular capillary wall.
e.g. nephrotic syndrome.
 Tubular proteinuria due to excretion of low molecular
weight proteins which are actively reabsorbed by proximal
renal tubules in diseased conditions of tubules.
e.g. acute and chronic pyelonephritis, heavy metal poisoning,
tuberculosis of kidney, interstitial nephritis, cystinosis, Fanconi
syndrome and rejection of kidney transplant.
 CHEMICAL EXAMINATION
Proteins
 Causes
 Overflow proteinuria: When concentration of a low molecular
weight protein rises in plasma, it “overflows” from plasma into
the urine.
e.g immunoglobulin light chains or Bence Jones proteins
(plasma cell dyscrasias), hemoglobin (intravascular
hemolysis), myoglobin (skeletal muscle trauma) &
lysozyme (acute myeloid leukemia type M4 or M5).
 Hemodynamic proteinuria: Alteration of blood flow through
the glomeruli causes increased filtration of proteins.
e.g high fever, hypertension, heavy exercise, congestive cardiac
failure, seizures,& exposure to cold.
 Post-renal proteinuria: This is caused by inflammatory or
neoplastic conditions in renal pelvis, ureter, bladder, prostate, or
urethra.
 CHEMICAL EXAMINATION
Test for Proteins
 Heat and acetic acid test
Principle –
Proteins are denatured &
coagulated upon heating to give
white cloud precipitate.
Method –
 Take a 5 ml test tube.
 Fill 2/3rd with urine.
 Acidify by adding a few drops
of 3% acetic acid if urine is
alkaline.
 Boil upper portion for 2 minutes
(lower part acts as control).
 If precipitation or turbidity appears, add a few drops of 3% acetic acid.
 CHEMICAL EXAMINATION
Test for Proteins
Heat and acetic acid test
Interpretation.
 If turbidity or precipitation
disappears on addition of acetic
acid, it is due to phosphates.
 if turbidity or precipitation
persists after addition of acetic
acid, then it is due to proteins.
 The test is semiquantitative and
can be graded from traces to 4+
depending upon amount of
protein.
 CHEMICAL EXAMINATION
Test for Proteins
 Sulphosalicylic Acid Test
Principle – Interpretation.
 Addition of sulphosalicylic acid
to the urine causes formation of a Appearance of turbidity
white precipitate if proteins are which persists after
present. heating indicates
Method – presence of proteins.
 Take 2 ml of clear urine in a test
tube.
 If reaction of urine is neutral or
alkaline, a drop of glacial acetic
acid is added.
 Add 2-3 drops of sulphosalicylic
acid (3 to 5%) and examine for
turbidity against a dark
background
 CHEMICAL EXAMINATION
Test for Proteins
 Heller’s Test
Method –
 Take 2 ml of concentrated nitric acid in a test
tube.
 Add urine drop by drop by the side of test tube.

Interpretation-
 Appearance of white ring at the junction indicates
presence of protein.
 CHEMICAL EXAMINATION
Test for Proteins
 Reagent Strip Method
Principle –
 The reagent area of the strip
is coated with bromophenol
blue indicator and buffered to
an acid pH which changes
color in the presence of
proteins.
Method –
 Bromophenol coated strip is Interpretation -
dipped in urine.
 Change in colour of strip
indicates presence of proteins
in urine and is compared with
the colour chart provided for
Reagent strip test is mainly reactive to albumin.
semiquantitative grading.
It is false-negative in the presence of Bence Jones proteins, myoglobin,
 CHEMICAL EXAMINATION
Microalbuminuria
 Urinary excretion of 30 to 300 mg/24 hours (or 2-20 mg/dl) of
albumin in urine.
 Significance –
 earliest sign of renal damage in diabetes mellitus (diabetic
nephropathy).
 independent risk factor for cardiovascular disease in diabetes
mellitus.
 Detection –
 Measurement of albumin-creatinine ratio in a random urine sample.
 Measurement of albumin in an early morning or random urine
sample.
 Measurement of albumin in a 24 hr sample .
 Test strips that screen for microalbuminuria are available
commercially.
 CHEMICAL EXAMINATION
Quantitative Estimation of Proteins in Urine.
Esbach’s albuminometer method
 Fill the albuminometer with urine up
to mark U.
 Add Esbach’s reagent (5g picric acid
+ 10g citric acid + 500ml of distilled
water) up to mark R
 Stopper the tube, mix it and let it
stand for 24 hours.
 Take the reading from the level of
precipitation in the albuminometer
tube and divide it by 10 to get the
percentage of proteins.
 CHEMICAL EXAMINATION
Quantitative Estimation of Proteins in Urine.

Turbidimetric method
 Take 1 ml of urine and 1 ml standard in two separate
tubes.
 Add 4 ml of trichloroacetic acid to each tube.
 After 5 minutes take the reading with red filter (680
nm).
 CHEMICAL EXAMINATION
Bence Jones Proteinuria

 Bence Jones proteins are monoclonal immunoglobulin

light chains (either κ or λ) that are synthesized by
neoplastic plasma cells.
 Excess production of these light chains occurs in plasma
cell dyscrasias like multiple myeloma and primary
amyloidosis.
 Because of their low molecular weight and high
concentration they are excreted in urine (overflow
proteinuria).
 CHEMICAL EXAMINATION
Test for Bence Jones Proteinuria
 Upon Δ, Bence Jones proteins precipitate at temperatures
between 40-60°C.
(other proteins precipitate between 60-70°C)
 The precipitate disappears on further heating at 85-100°C.
(while precipitate of other proteins does not)
 When cooled (60-85°C), there is reappearance of
precipitate of Bence Jones proteins.
•This test is not specific for Bence Jones proteins and both false-positive
and negative results can occur.
• This test has been replaced by protein electrophoresis of concentrated
urine sample.
• Protein electrophoresis – Movement of charged particles through an
electrolyte subjected to an electric field. e.g. M band in multiple myeloma
 CHEMICAL EXAMINATION
Glucose
 Main indication for testing glucose in urine is detection
of unsuspected diabetes mellitus or follow-up of
known diabetic patients.
 All of the glucose filtered by the glomeruli is
reabsorbed by the proximal renal tubules and
returned to circulation.
 Normally a very small amount of glucose is excreted
in urine that cannot be detected by the routine tests.
(< 500 mg/24 hours or <15 mg/dl)
 Presence of detectable amounts of glucose in urine is
glycosuria.
 Glycosuria results if the filtered glucose load exceeds
the capacity of renal tubular reabsorption.
 CHEMICAL EXAMINATION
Glucose
Causes of Glycosuria
Glycosuria with hyperglycemia:
 Endocrine diseases: diabetes mellitus, acromegaly, Cushing’s
syndrome, hyperthyroidism, pancreatic disease.
 Non-endocrine diseases: central nervous system diseases, liver
disorders.
 Drugs: adrenocorticotrophic hormone, corticosteroids, thiazides.
 Alimentary glycosuria (Lag-storage glycosuria):
 After a meal, there is rapid intestinal absorption of glucose leading to
transient elevation of blood glucose above renal threshold.
 This can occur in persons with gastrectomy or gastrojejunostomy
and in hyperthyroidism.
 Glucose tolerance test reveals a peak at 1 hour above renal threshold
(which causes glycosuria); the fasting and 2-hour glucose values are
normal.
 CHEMICAL EXAMINATION
Glucose
Causes of Glycosuria
Glycosuria without hyperglycemia: Renal glycosuria
 This accounts for 5% of cases of glycosuria in general
population.
 Renal threshold is the highest glucose level in blood at which
glucose appears in urine and which is detectable by routine
laboratory tests.
 The normal renal threshold for glucose is 180 mg/dl.
 The renal threshold is set below 180 mgs/dl but glucose
tolerance is normal
 The disorder is transmitted as autosomal dominant.
 CHEMICAL EXAMINATION
Test for Glucose
Benedict’s qualitative test:
Composition of Benedict’s qualitative reagent:
Copper sulphate 17.3 gram
Sodium carbonate 100 gram
Sodium citrate 173 gram
Distilled water 1000 ml

Principle –
 When urine is boiled in Benedict’s qualitative solution,
blue alkaline copper sulphate is reduced to red-brown
cuprous oxide if abreducing agent is present.
 CHEMICAL EXAMINATION
Test for Glucose
Benedict’s qualitative test:
Methods: Interpretation:
 The result is reported in grades
 Add 0.5 ml (or 8 drops) of as follows :
urine. Mix well.  Nil: no change from blue color
 Boil over a flame for 2 minutes.  Trace: Green without
 Allow to cool at room
precipitate
temperature.  1+ (approx. 0.5 grams/dl):
Green with precipitate
 Note the color change, if any.  2+ (approx. 1.0 grams/dl):
Brown precipitate
 3+ (approx. 1.5 grams/dl:
Yellow-orange precipitate
 4+ (> 2.0 grams/dl): Brick- red
precipitate.
 CHEMICAL EXAMINATION
Test for Glucose
Clinitest tablet method (Copper reduction tablet test):
 This is a modified form of Benedict’s test in which the
reagents are present in a tablet form.
 Sensitivity is 200 mgs/dl of glucose.
 CHEMICAL EXAMINATION
Test for Glucose
Reagent strip method
 This test is specific for glucose and is therefore preferred over
Benedict’s and Clinitest methods.
 It is based on glucose oxidase-peroxidase reaction.
 Reagent area of the strips is impregnated with two enzymes - glucose
oxidase and peroxidase and a chromogen.

 Nature of chromogen and buffer system differ in different strips.

 The strip is dipped into the urine sample and color is observed after a
specified time and compared with the color chart provided.
 This test is more sensitive than Benedict’s qualitative test and specific
only for glucose. Other reducing agents give negative reaction.
 CHEMICAL EXAMINATION
Ketones
 Excretion of ketone bodies (acetoacetic acid, β-
hydroxybutyric acid, and acetone) in urine is called
as ketonuria.
 Ketones are breakdown products of fatty acids and
their presence in urine is indicative of excessive fatty
acid metabolism to provide energy.
 Normally ketone bodies are not detectable in the urine
of healthy persons.
 If energy requirements cannot be met by metabolism
of glucose (due to defective carbohydrate
metabolism, low carbohydrate intake, or increased
metabolic needs), then energy is derived from
breakdown of fats formation of ketone bodies
 CHEMICAL EXAMINATION
Causes of Ketonuria
Decreased utilization of carbohydrates
 Uncontrolled diabetes mellitus with ketoacidosis
compensatory increased lipolysis  increase in the level
of free fatty acids in plasma.
 Degradation of free fatty acids in the liver  the formation
of acetoacetyl CoA which then forms ketone bodies.
 Ketone bodies are strong acids and produce H+ ions,
which are neutralized by bicarbonate ions.
 Fall in bicarbonate (i.e. alkali) level produces ketoacidosis.
 Ketone bodies also increase the plasma osmolality and
cause cellular dehydration.
 Presence of ketone bodies in urine may be a warning of
impending ketoacidotic coma
 Glycogen storage disease (von Gierke’s disease)
 CHEMICAL EXAMINATION
Causes of Ketonuria
 Decreased availability of carbohydrates in the diet:
1. Starvation
2. Persistent vomiting in children
3. Weight reduction program (severe carbohydrate
restriction with normal fat intake)

 Increased metabolic needs:

 a. Fever in children
 b. Severe thyrotoxicosis
 c. Pregnancy
 d. Protein calorie malnutrition
 CHEMICAL EXAMINATION
Tests For Ketone Bodies
Rothera’s Test
Principle :
Acetoacetic acid or acetone reacts with nitroprusside in alkaline
solution to form a purple-colored complex.

Method:
 Take 5 ml of urine in a test tube and saturate
it with ammonium sulphate.
 Add a small crystal of sodium nitroprusside.
Mix well.
 Slowly run along the side of the test tube
liquor ammonia to form a layer.
 Immediate formation of a purple
permanganate colored ring at the junction of
the two fluids indicates a positive test
 CHEMICAL EXAMINATION
Tests For Ketone Bodies
Acetest tablet test

This is Rothera’s test in the form of a tablet.

The test is more sensitive than reagent strip
test for ketones.
The Acetest tablet consists of sodium nitroprusside, glycine, and an
alkaline buffer.
A purple lavender discoloration of the tablet indicates the presence
of acetoacetate or acetone (≥ 5 mg/dl).
A rough estimate of the amount of ketone bodies can be obtained
bybcomparison with the color chart provided by the manufacturer
 CHEMICAL EXAMINATION
Tests For Ketone Bodies
Ferric chloride test (Gerhardt’s)
 Addition of 10% ferric chloride solution to urine
causes solution to become reddish or purplish if
acetoacetic acid is present.
 The test is not specific since certain drugs
(salicylate and L-dopa)give similar reaction.
 Sensitivity of the test is 25-50 mg/dl.
 CHEMICAL EXAMINATION
Tests For Ketone Bodies
Reagent strip test
 Reagent strips tests are modifications of
nitroprusside test.
 Their sensitivity is 5-10 mg/dl of acetoacetate.
 If exposed to moisture, reagent strips often give
false-negative result.
 Ketone pad on the strip test is especially
vulnerable to improper storage and easily gets
damaged.
 CHEMICAL EXAMINATION
Bile Pigment (Bilirubin)
 Bilirubin (a breakdown product of hemoglobin) is
undetectable in the urine of normal persons.
 Presence of bilirubin in urine is called as
bilirubinuria.
After its formation from
hemoglobin in
reticuloendothelial
system
Unconjugated bilirubin is not water-
soluble, is bound to albumin, and cannot
pass through the glomeruli. Therefore, it
does not appear in urine

Conjugated bilirubin is water soluble, is

filtered by the glomeruli, and therefore
appears in urine.
 CHEMICAL EXAMINATION
Bile Pigment (Bilirubin)
Detection of bilirubin in urine (along with urobilinogen)
is helpful in the differential diagnosis of jaundice
Urine test Hemolytic Hepatocellular Obstructive
Jaundice Jaundice Jaundice

Bilirubin Absent Present Present

Urobilinogen Increased Increased Absent

•In acute viral hepatitis, bilirubin appears in urine even before jaundice is
clinically apparent.

•Presence of bilirubin in urine indicates conjugated hyperbilirubinemia

(obstructive or hepatocellular jaundice).
 CHEMICAL EXAMINATION
Tests For Detection of Bilirubin in Urine
1. Foam test:
 About 5 ml of urine in a test tube is shaken and observed for
development of yellowish foam.
 Similar result is also obtained with proteins and highly
concentrated urine.
 In normal urine, foam is white.

2. Gmelin’s test:
 Take 3 ml of concentrated nitric acid in a test tube and slowly
placetube
• The equal quantitygently;
is shaken of urine over
play of it.
colors
(yellow, red, violet, blue, & green) indicates
+ve test.
3. Lugol iodine test:
Take 4 ml of Lugol iodine solution (Iodine 1 gm, potassium
iodide 2 gm, and distilled water to make 100 ml) in a test
tube and add 4 drops of urine. Mix by shaking. Development
of green color indicates positive test.
 CHEMICAL EXAMINATION
Tests For Detection of Bilirubin in Urine
4. Fouchet’s test: A simple and sensitive test.
i. Add 2.5 ml of 10% of barium chloride to 5 ml of fresh urine in a
test tube and mix well. A precipitate of sulphates appears to which
bilirubin is bound (barium sulphate-bilirubin complex).
ii. Filter to obtain the precipitate on a filter paper.
iii. To the precipitate on the filter paper, add 1 drop of Fouchet’s
reagent (25 g of trichloroacetic acid, 10 ml of 10% ferric chloride,
and distilled water 100 ml).
iv. Immediate development of blue-green color around the drop
indicates presence of bilirubin.
 CHEMICAL EXAMINATION
Tests For Detection of Bilirubin in Urine

5. Reagent strips or tablets impregnated with diazo

reagent:
 These tests are based on reaction of bilirubin with diazo
reagent.
 The color change is proportional to the concentration of
bilirubin.
 Tablets (Ictotest) detect 0.05-0.1 mg of bilirubin/dl of urine.
 The reagent strip tests are less sensitive (0.5 mg/dl).
 CHEMICAL EXAMINATION
Bile Salts
 Bile salts are salts of bile acids: cholic, deoxycholic,
chenodeoxycholic, and lithocholic.
 These bile acids combine with glycine or taurine to form
complex salts or acids.
 Bile salts enter the small intestine through the bile and act
as detergents to emulsify fat and reduce the surface tension
on fat droplets so that enzymes (lipases) can breakdown the
fat.
 In the terminal ileum, bile salts are absorbed and enter in
the blood stream from where they are taken up by the liver
and re-excreted in bile (enterohepatic circulation).
 CHEMICAL EXAMINATION
Bile Salts
 Bile salts along with bilirubin can be detected in
urine in cases of obstructive jaundice.
 Bile salts and conjugated bilirubin regurgitate into
blood from biliary canaliculi (due to increased
intrabiliary pressure) and are excreted in urine.
 The test used for the detection of bile salts is
Hay’s surface tension test.
 The property of bile salts to lower the surface
tension is utilized in this test.
 CHEMICAL EXAMINATION
Tests For Bile Salts
Hay’s surface tension test :
 Take some fresh urine in a conical glass tube.
 Urine should be at the room temperature.
 Sprinkle on the surface particles of sulphur.
 If bile salts are present, sulphur particles sink to
the bottom because of lowering of surface tension
by bile salts.
 If sulphur particles remain on the surface of urine,
bile salts are absent.
 Thymol (used as a preservative) gives false
positive test.
 CHEMICAL EXAMINATION
Urobilinogen
 Conjugated bilirubin excreted into the duodenum through bile is
converted by bacterial action to urobilinogen in the intestine.
 Major part is eliminated in the feces.
 A portion of urobilinogen is absorbed in blood, which
 undergoes recycling (enterohepatic circulation)
 A small amount, which is not taken up by the liver, is excreted in
urine.
 Urobilinogen is colorless; upon oxidation it is converted to
urobilin, which is orange-yellow in color.
 Normally about 0.5-4 mg of urobilinogen is excreted in
urine in 24 hours.
 Urinary excretion of urobilinogen shows diurnal variation
with highest levels in afternoon.
 A 2-hour post-meal sample is usually preferred.
 CHEMICAL EXAMINATION
Urobilinogen
 Conjugated bilirubin excreted into the duodenum through bile is
converted by bacterial action to urobilinogen in the intestine.
 Major part is eliminated in the feces.
 A portion of urobilinogen is absorbed in blood, which
 undergoes recycling (enterohepatic circulation)
 A small amount, which is not taken up by the liver, is excreted in
urine.
 Urobilinogen is colorless; upon oxidation it is converted to
urobilin, which is orange-yellow in color.
 Normally about 0.5-4 mg of urobilinogen is excreted in
urine in 24 hours.
 Urinary excretion of urobilinogen shows diurnal variation
with highest levels in afternoon.
 A 2-hour post-meal sample is usually preferred.
 CHEMICAL EXAMINATION
Urobilinogen
Causes of Increased Urobilinogen in Urine
1. Hemolysis
 Increased urobilinogen in urine without bilirubin
 Excessive destruction of red cells leads to hyperbilirubinemia and
therefore increased formation of urobilinogen in the gut.
2. Hemorrhage in tissues:
 There is increased formation of bilirubin from destruction of red
cells.
Causes of Reduced Urobilinogen in Urine
1. Obstructive jaundice:
In biliary tract obstruction,delivery of bilirubin to the intestine is restricted
and
very Reduction
2. little or no urobilinogen
of intestinalisbacterial
formedclay-colored
flora: stools.
This prevents conversion of bilirubin to urobilinogen in the intestine.
It is observed in neonates and following antibiotic treatment
 CHEMICAL EXAMINATION
Tests For Urobilinogen
Ehrlich’s aldehyde test
 Ehrlich’s reagent (p-dimethylaminobenzaldehyde) reacts with
urobilinogen in urine to produce a pink color.
 Intensity of color developed depends on the amount of urobilinogen
present.
 Take 5 ml of fresh urine in a test tube.
 Add 0.5 ml of Ehrlich’s aldehyde reagent (which consists of
ydrochloric acid 20 ml, distilled water 80 ml, and
paradimethylaminobenzaldehyde 2 gm).
 Allow to stand at room temperature for 5 minutes.
 Development of pink color indicates normal amount of
urobilinogen.
 Dark red color means increased amount of urobilinogen.
 CHEMICAL EXAMINATION
Tests For Urobilinogen
Watson-Schwartz Test
 Distinguish between both urobilinogen and porphobilinogen.
 Add 1-2 ml of chloroform, shake for 2 minutes and allow to
stand.
 Pink color in the chloroform layer indicates presence of
urobilinogen, while pink coloration of aqueous portion indicates
presence of porphobilinogen.

 False-negative reaction can occur in the presence of

(i) urinary tract infection (nitrites oxidize urobilinogen to urobilin)
(ii) antibiotic therapy (gut bacteria which produce urobilinogen are
destroyed).
 CHEMICAL EXAMINATION
Tests For Urobilinogen
Reagent strip method:
 This method is specific for urobilinogen.
 Test area is impregnated with either p-
dimethylaminobenzaldehyde or 4-methoxybenzene
diazonium tetrafluoroborate.
 CHEMICAL EXAMINATION
Blood
 The presence of abnormal number of intact red blood cells in urine is
called as hematuria.
Causes of Hematuria
1. Diseases of urinary tract
• Glomerular diseases: Glomerulonephritis, Berger’sdisease, lupus
nephritis, Henoch-S.chonlein purpura.
 Non-glomerular diseases: Calculus, tumor, infection,
tuberculosis, pyelonephritis, hydronephrosis, polycystic kidney
disease, trauma, after strenuous physical exercise, diseases of
prostate (benign hyperplasia of prostate, carcinoma of prostate.
2. Hematological conditions:
 Coagulation disorders, sickle cell disease
 CHEMICAL EXAMINATION
Tests for Detection of Blood in Urine
1. Microscopic examination of urinary sediment:
 Definition of microscopic hematuria is presence of 3 or more number of red
blood cells per high power field on microscopic examination of urinary
sediment in two out of three properly collected samples.
2. Chemical tests:
 Benzidine test:
 Make saturated solution of benzidine in glacial acetic acid.
 Mix 1 ml of this solution with 1 ml of hydrogen peroxide in a test tube.
 Add 2 ml of urine.
 If green or blue color develops within 5 minutes, the test is positive.
 Orthotoluidine test:
 In this test, instead of benzidine, orthotoluidine is used.
 It is more sensitive than benzidine test.
 Reagent strip test:
 Various reagent strips are commercially available which use different chromogens (o-
toluidine, tetramethylbenzidine).
 CHEMICAL EXAMINATION
Tests for Detection of Blood in Urine
Chemical tests are positive in hematuria, hemoglobinuria and myoglobinuria.
 CHEMICAL EXAMINATION
Chemical Tests for Significant
Bacteriuria
1. Nitrite test:
 Nitrites are not present in normal urine.
 Ingested nitrites are converted to nitrate and excreted in urine.
 If gram-negative bacteria (e.g. E.coli,Salmonella, Proteus, Klebsiella,
etc.) are present in urine
 Nitrites are then detected in urine by reagent strip tests.

2. Leucocyte esterase test:

 It detects esterase enzyme released in urine from granules of
leucocytes.
 Thus the test is positive in pyuria. If this test is positive, urine
culture should be done.
 The test is not sensitive to leucocytes < 5/HPF.
 URINE EXAMINATION

 PHYSICAL EXAMINATION

 CHEMICAL EXAMINATION

 MICROSCOPIC EXAMINATION
 MICROSCOPIC EXAMINATION
 Normal urine microscopy contains few epithelial cells,
occasional RBC’s, few crystals.
 Urine consists of various microscopic, insoluble, solid
elements in suspension.
 These elements are classified as organized or
unorganized.
 Organized substances include red blood cells, white
blood cells, epithelial cells, casts, bacteria, and parasites.
 Unorganized substances are crystalline and amorphous
material which are suspended in urine and on standing
they settle down and sediment at the bottom of the
containerurinary deposits or urinary sediments.
The cellular elements are best preserved in acid,
hypertonic urine; they deteriorate rapidly in alkaline,
hypotonic solution.
 MICROSCOPIC EXAMINATION
 Microscopic urinalysis is
done by pouring the urine
sample into a test tube
and centrifuging it for a
few minutes.
 The top liquid part (the
supernatant) is discarded.
 The solid part left in the
bottom of the test tube
(the urine sediment) is
mixed with the remaining
drop of urine in the test
tube
 one drop of this is
analyzed under a
microscope
MICROSCOPIC EXAMINATION
 MICROSCOPIC EXAMINATION
Urinary findings in renal diseases
MICROSCOPIC EXAMINATION
Cells in urine
 MICROSCOPIC EXAMINATION
Crystals
 Crystals are refractile
structures with a definite
geometric shape due to
orderly 3-dimensional
arrangement of its
atoms and molecules.
 Amorphous material (or
deposit) has no definite
shape and is commonly
seen in the form of
granular aggregates or
clumps
MICROSCOPIC EXAMINATION
Crystals
 MICROSCOPIC EXAMINATION
Crystals
Crystals in acidic urine Crystals in alkaline urine
 Uric acid  Ammonium magnesium

 Calcium oxalate phosphates(triple

phosphate crystals)
 Cystine
 Calcium carbonate
 Leucine
 Amorphous phosphates
 Ammonium urate crystals
 MICROSCOPIC EXAMINATION
Casts in urine
 Urinary casts are cylindrical aggregations of particles that form in the distal
nephron, dislodge and pass in the urine.
 Casts are of two main types:
 Noncellular: Hyaline, granular, waxy, fatty
 Cellular: Red blood cell, white blood cell, renal tubular epithelial cell.
 MICROSCOPIC EXAMINATION
Casts in urine
Hyaline casts
 Most common type of
casts which are
composed of solidified
Tamm-Horsfall
mucoprotein.
 They have smooth
texture and a
They may be seen in healthy patients,
refractive index very increased numbers during dehydration,
close to that of the exercise or diuretic medicines.
surrounding fluid.
MICROSCOPIC EXAMINATION
Casts in urine
Granular Casts
 Granular casts result
either from the
degeneration of
cellular casts, or direct
aggregation of plasma
proteins or
immunoglobulin light
chains.
 They have a textured They are seen after sternous exercise,
appearance which chronic renal diseases, acute tubular
ranges from fine to necrosis etc.
coarse in character.
MICROSCOPIC EXAMINATION
Casts in urine
Waxy Casts

 Waxy casts
represent the final
stage of
degeneration of
cellular casts. They
are more refractile
seen in tubular injury of a more chronic
nature than granular or cellular casts like
severe chronic renal disease and renal
amyloidosis. These casts are also
called renal failure casts.
 MICROSCOPIC EXAMINATION
Casts in urine
Fatty Casts

 Fatty casts are

formed by the
breakdown of lipid-
rich epithelial cells.
These contain lipid
droplets within the
protein matrix of the
cast and are
identified by the
presence of refractile
They are usually seen in the conditions like
lipid droplets.
tubular degeneration, nephrotic syndrome,
 hypothyroidism etc.
 MICROSCOPIC EXAMINATION
Casts in urine
Red blood cell casts

 The presence of
red blood cells
within the cast is
always pathologic,
and is strongly
indicative of
glomerular
damage.
 They are usually
associated with
nephritic
syndromes.
 MICROSCOPIC EXAMINATION
Casts in urine
White blood cell casts

 White blood cells

(generally
neutrophils) are
present within or
upon casts.
 Indicative of
inflammation or
infection.
 These casts are
typical for acute
pyelonephritis
 MICROSCOPIC EXAMINATION
Casts in urine
Renal Tubular Epithelial Cell Casts

 These casts are

composed of renal
epithelial cells.
 These casts are
seen in conditions
such as renal
tubular necrosis,
viral disease (such
as CMV nephritis),
and kidney
transplant
MICROSCOPIC EXAMINATION
Organisms in urine
MICROSCOPIC EXAMINATION
Organisms in urine