Clinical Pathology 1 part

st
Department of Pathology
 Clinical pathology
It is the part of pathology, which assist clinician in concluding
the diagnosis, which involves the examination of specimens
collected from the patients which includes:
Urine
Stool
Sputum
Blood
CSF
Other Body Fluids
Biopsy Material.
Competency (PA – 23.1)
Describe abnormal urinary findings in disease states and identify and
describe common urinary abnormalities in a clinical specimen
1.

1. Identify and describe abnormal physical characteristics accurately in

different diseases in urine
2. Identify and describe abnormal chemical constituents of urine correctly in
a disease state in a clinical specimen.
3. Perform qualitative and semiquantitative laboratory tests to detect
common abnormal chemical constituents of urine in a diseased state in a
clinical specimen correctly
4. Interpret the results of these tests and discuss their clinical relevance with
reference to a given set of numerical parameters.
5. Identify and interpret abnormal microscopic findings correctly in various
disease state in a urine sample
6. Correlate macroscopic and microscopic urinary findings in a clinical
specimen correctly.
Specific learning objectives
1. Composition of normal urine
2. Indications for urinalysis
3. Collection of urine and its methods
4. Cautions in sample collection
5. Changes that occur in standing urine
6. Urinalysis examination
I. Physical examination
II. Chemical analysis
III. Microscopic examination
 Why do Urinalysis
Fluid biopsy of the kidneys
Non – invasive, cost effective
Urine is an ultra filtrate of the plasma, it can be
used to evaluate and monitor body
homeostasis and many metabolic disease
processes.
1.Composition of normal urine
2. Indications of 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.
3.Types of Urine Collection (Sampling)
3. Urine Collection Methods :Clean catch
midstream sample
External genitalia is cleaned and midstream void taken for testing
urine for microorganisms or presence of infection
3. Urine Collection Methods: Pediatric
Sample
External genitalia cleaned
Special urine collection bag
is adhered to the skin
surrounding the urethral
area.
The urine is transferred into
an evacuated tube
3. Urine Collection Methods: Supra-pubic Needle Aspiration

This method is used in

bedridden patient cannot be
catheterized or a sterile
specimen is required.
The urine specimen is
collected by needle
aspiration through the
abdominal wall into the
bladder.
3. Urine Collection Methods: Catheter
collection sample
This assisted procedure is
conducted when a patient is
bedridden or cannot urinate
independently.
Foley catheter inserts into the
bladder through the urethra to
collect the urine specimen.
4. Caution in sample collection
→ Should avoid athletic or heavy physical work
→ Sample tested within one hour of collection or
refrigerated.
→ For chemical tests , urine can be stored at 2–
8°C for 24 hours and microscopic examination
within 4 hours
4. Caution in sample collection (cont):
Preservation
After 24 hours collection urine sample use chemical
preservation :-
Toluene → Doesn’t interfere with routine analyses
Thymol → Preserves glucose and sediments well
Boric acid → Preserves protein and doesn’t interfere
with routine analyses
Formalin → preserve cells and casts
5. Changes that occur in standing urine
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.
 6. Urinalysis examination
1) Physical examination of urine
1.

1.

1. Volume – measurement and interpretation

2. Colour – identification and interpretation with clinical
correlation
3. Appearance – various causes of turbid urine and their
clinical importance
4. Odour – abnormal odour and their clinical correlation
5. Specific Gravity –different ways to measure and variation
in disease states
6. Ph – different ways to measure and variation in disease
states
I. Physical examination of urine 1) Volume (cont)

The average adult : 1200ml to 2000ml per day

Increase
Polyuria ---more than 2000ml of urine in 24 hours
1. Physiological states:
states water intake, some drugs,
intravenous solutions
2. Pathologic states: Diabetes mellitus, Diabetes Insipidus
I. Physical examination of urine 1) Volume (cont)

Decrease
Oliguria ---less than 400ml of urine in 24 hours
Anuria ---less than 100ml of urine in 24 hours
1) Prerenal: hemorrhage, dehydration, congestive heart failure
2) Postrenal: obstruction of the urinary tract

(may be stones, carcinoma)

3) Renal parenchymal disease:
Acute tubular necrosis, Chronic renal failure
I. Physical examination of urine2) Colour
Visual examination Including color and clarity
Normal Colour : pale to dark yellow (urochrome)
Abnormal color :
- Physiological : Some drugs cause color changes
- Pathological :
1. Red urine →Hematuria
→
Cause – Hemoglobinuria, Porphyria
2. Yellow-brown or Green-brown Urine → Bilirubin
Cause -obstructive jaundice
 I. Physical examination of urine2)
Colour (cont)
3. Brown Urine → Prophobilin or Urobilin
Examples of Urine Color
I. Physical examination of urine3)
Appearance
Normal clarity: Clear
Abnormal clarity: Cloudy urine
Examples of Urine Clarity
I. Physical examination of urine 4) Odour
Aromatic → Normal
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
I. Physical examination of urine 5) Specific
gravity (Relative Mass Density)
Specific gravity is a measure of concentrating ability of
kidneys and its tubular function
It depends on amount of solutes in urine solution.
It is a comparison of density of urine against the density
of distilled water at a particular temperature
Normal Specific gravity of urine is 1.016 – 1.022
Specific gravity increases as solute concentration
increases and decreases when temperature rises ( since
volume expands with rise in temperature)
I. Physical examination of urine 5) Specific gravity
(cont)
Hypersthenuria (↑ specific gravity)
Diabetes mellitus
Glycosuria
Albuminuria
Fever and dehydration
Hyposthenuria (↓ specific gravity)
Diabetes insipidus
Pyelonephritis
Diuretics
Alcohol
Isosthenuria – low and fixed SG at 1.010 due to loss of
concentrating ability of tubules seen in end stage renal failure
I. Physical examination: 5) Specific
Gravity(cont) Urinometer
This method is base on the principle of buoyancy (the ability of a
fluid to exert an upward thrust on a body placed in it).
5) Specific gravity – Refractometer methodRefractometer
precisely measures the refractive index of the total soluble solids
5) Specific gravity –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 colour of pH
indicator (bromothymol blue)
I. Physical examination: 6) Urine 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 CO 2 and
production of ammonia from urea.
pH normal value – 4.6 – 8
Urine pH depends on the diet, acid base balance, water
balance and renal tubular function.
I. Physical examination: 6) Urine pH (cont)
Higher PH---alkaline urine
1) Drugs: sodium bicarbonate
2) Classic renal tubular acidosis
3) Alkalosis (metabolic or respiratory)
Lower PH---acid urine
1) Drugs: Ammonium Chloride
2) Acidosis (Metabolic Or Respiratory)
Various methods for determination of urine :
Litmus paper
pH indicator paper
pH meter
Reagent strip tests
Methods for determination of urine pH
Methods for determination of urine pH
3. pH meter:
An electrode of pH meter is dipped in urine sample and pH is read off
directly from digital display
It is used if exact pH is required
Methods for determination of urine pH
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.
Methods for determination of urine pH
4. Reagent strip test:
Reagent strips are used only once and discarded.
6. Urinalysis examination II. Chemical analysis
The chemical examination is carried out for the following
substance:
6. Urinalysis examination II. Chemical analysis
1) Protein: The presence of protein in urine (Proteinuria or
Albuminuria) can be seen in patients with
glomerulonephritis
Proteinuria refers to protein excretion in urine greater
than 150 mg/24 hours in adults.
Proteinuria (depend on the amount of protein )
Heavy proteinuria ＞ 3.5g/24 hours
Moderate proteinuria = 1.0-3.5g/24 hours
Minimal proteinuria ＜ 1.0g/24 hours
 II. Chemical analysis 1) Protein
Causes
1. Glomerular proteinuria :- ↑permeability of glomerular capillary wall
e.g. Nephrotic syndrome
2. Tubular proteinuria :- excretion of low molecular weight proteins in
disease conditions of tubules (actively reabsorbed by proximal renal tubules)
e.g.
Acute and chronic pyelonephritis
Heavy metal poisoning
Tuberculosis of kidney
Interstitial nephritis
Cystinosis
Fanconi syndrome
Rejection of kidney transplant
II. Chemical analysis 1) Protein (cont)
3. Overflow proteinuria:
When concentration of a low molecular weight protein rises in plasma,
it “overflow” from plasma into the urine.
Eg: - Immunoglobulin Light Chain or Bence Jones Proteins (Plasma
Cell Dyscrasias),
4. Hemoglobin (intravascular hemolysis),
Myoglobin (skeletal muscle trauma) and
lysozyme (acute myeloid leukemia type M4 or M5)
II. Chemical analysis 1) Protein (cont)
5. Hemodynamic proteinuria:
Alteration of blood flow through the glomeruli causes increased
filtration of proteins.
e.g
High fever,
Hypertension
Heavy exercise
Congestive cardiac failure
Exposure to cold.
6. Post renal proteinuria:
This is caused by inflammatory or neoplastic conditions in renal pelvis,
ureter, bladder, prostate or urethra
 II. Chemical analysis 1) Test For Protein (cont)
a) Heat and acetic acid test
Principle-
Protein are denatured and coagulated upon heating to give white cloud
precipitate.
Method
Take a 5ml 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.
II. Chemical analysis 1) Test For Protein (cont)
a) 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.
 II. Chemical analysis 1) Test For Protein (cont)
b) Sulphosalicylic acid test
Principle –
Addition of sulphosalicylic acid to the
urine causes formation of a white
precipitate if proteins are present.
Method –
Take 2ml of clear urine is neutral or alkaline,
a drop of glacial acetic acid is added.
If reaction of urine is neutral or alkaline, a
drop of glacial acid is added. Interpretation.
Add 2-3 drops of sulphosalicylic acid (3 to Appearance of turbidity
5%) and examine for turbidity against a dark which persists after
heating indicates
background presence of proteins
 II. Chemical analysis 1) Test For Protein (cont)
c) 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.
 II. Chemical analysis 1) Test For Protein (cont)
d) Reagent strip test
The principle is known as “protein error of
indicators”.
The reagent area is impregnated with
bromophenol blue indicator buffered to pH 3.0
with citrate.
When the dye gets adsorbed to protein, there is
change in ionization (and hence pH) of the
indicator that leads to change in color of the
indicator.
The intensity of the color produced is
proportional to the concentration of protein.
e) Quantitative estimation of proteins in urine
Indications for quantitative estimation of proteins in urine are:
Diagnosis of nephrotic syndrome
Detection of microalbuminuria or early diabetic nephropathy
To follow response to therapy in renal disease
Methods include :
1. Estimation of proteins in a 24-hour urine sample
2. Estimation of protein/creatinine ratio in a random urine sample.
Clinical significance :
Proteinuria >1500 mg/ 24 hours indicates glomerular disease
Proteinuria >3500 mg/24 hours is called as nephrotic range proteinuria; in
tubular, hemodynamic and post renal diseases,
Proteinuria is usually < 1500 mg/ 24 hours.
 Protein: 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.
 e) Quantitative estimation of proteins in urine
1. 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
e) Quantitative estimation of proteins in
urine
2. 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).
II. Chemical analysisBence
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).
II. Chemical analysis Test for Bence Jones
Proteinuria
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 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
II. Chemical analysis b) Glucose
Glycosuria :-Presence of glucose in urine
The main indication for testing for glucose in urine is detection of
unsuspected diabetes mellitus or follow-up of known diabetic patients
Causes
1) Glycosuria with Hyperglycemia:
1. Endocrine diseases: Diabetes Mellitus, Acromegaly, Cushing’s
Syndrome, Hyperthyroidism, Pancreatic Disease.
2. Non-endocrine diseases: Central Nervous System Diseases, Liver
Disorders.
3. Drugs: Adrenocorticotrophic Hormone, Corticosteroids, Thiazides.
b) Glycosuria - Causes
4. 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.
Glycosuria – Causes (cont)
5. 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.
II. Chemical analysis : Test for Glucose
1) 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 a reducing
agent is present
 1) Benedict’s qualitative test: (cont)
Methods: Interpretation:
Add 0.5 ml (or 8 drops) of urine to The result is reported in grades as follows :
5ml of Benedict’s solution.
Mix well and boil over a flame Nil: no change from blue color
for 2 minutes. Trace: Green without precipitate
Allow to cool at room 1+ (approx. 0.5 grams/dl):
temperature. 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.
II. Chemical analysis Test for Glucose
2. 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.
II. Chemical analysis : b)Test for Glucose
3. Reagent strip method
This test is specific for glucose and is therefore preferred over
Benedict’s and Clinitest methods. It is a double sequential enzyme
reaction
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.

II. Chemical analysisc) Ketone
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 before formation of ketone bodies
II. Chemical analysis causes for Ketone
1. Decreased utilization of carbohydrates
Uncontrolled diabetes mellitus with ketoacidosis→ compensatory increased
lipolysis→ increase in the levels 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 acid and produce H+ ions, which are neutralized by
bicarbonate ions.
Fall in bicarbonate (i.e, alkali) level produce ketoacidosis.
Ketone bodies also increase in 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)
II. Chemical analysis : causes for Ketonuria
2. Decreased availability of carbohydrates in the diet:
- Starvation
- Persistent vomiting in children
- Weight reduction diet (severe carbohydrate restriction with normal fat
intake)
3. Increased metabolic needs:
- Fever in children
- Severe thyrotoxicosis
- Pregnancy
- Protein calorie malnutrition
 II. Chemical analysis c)Tests for Ketone bodies
1. 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
 II. Chemical analysisc)Tests for Ketone bodies
2. 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
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
by comparison with the color chart provided by the manufacturer
II. Chemical analysisc)Tests for Ketone
bodies
3. 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.
II. Chemical Examination: d) Bile Pigment (Bilirubin)

Bilirubin (a breakdown product of hemoglobin) is not

detectable in the urine normally.
Presence of bilirubin in urine is called as Bilirubinuria
Unconjugated bilirubin is water insoluble and cannot pass
through the glomerulus
Conjugated bilirubin is water soluble and indicates further
evaluation for liver dysfunction and biliary obstruction when it
is detected in the urine.
Hemolysis and hepatocellular disease can elevate urobilinogen
levels, and antibiotic use and bile duct obstruction can decrease
urobilinogen levels.
II. Chemical analysis d) Bile Pigment (Bilirubin)
Detection of bilirubin in urine (along with urobilinogen) is helpful in the
differential diagnosis of jaundice

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).
II. Chemical analysis : d) Tests for detecting
Bilirubin
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.
 II. Chemical analysis : d)Tests for detecting Bilirubin
2) Gmelin’s test:
Take 3ml of concentrated nitic acid in a test
tube and slowly place equal quantity of urine
over it.
The tubes are shaken gently play for colors
(yellow, red, violet, blue and green) indicates
test is positive
II. Chemical analysis : d)Tests for
detecting Bilirubin
3) Lugol iodine test:
Take 4 ml of Lugol Iodine solution (Iodine 1gm, potassium
iodine 2gms and distilled water to make 100ml) in a test tube
and add 4 drops of urine. Mix by shaking. Development of
green color indicates positive test.
 II. Chemical analysisd) Tests For Detection of Bilirubin
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.
ii. Filter through filter paper to obtain the precipitate.
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.
II. Chemical analysis d)Tests for detecting
Bilirubin
5) Reagent strips or tablets impregnated with diazo
reagent:
These tests are based on reaction of bilirubin with diazo
reagent.
This test is based on the coupling of bilirubin with
diazotized dichloroaniline in a strongly acid medium
resulting in the development of various shades of tan
colour.
II. Chemical analysise) Bile Salts
Bile salts are salts of bile acids: cholic, 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)
II. Chemical analysise)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.
II. Chemical analysis d) Test to detect Bile Salts
1. 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.
II. Chemical analysis f) 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 is excreted in urine.
Urobilinogen is colorless; upon oxidation it is converted to
urobilin, which is orange-yellow in color.
Normally 0.5-4 mg excreted in urine in 24 hours.
II. Chemical analysis f) Urobilinogen
Causes of Increased Urobilinogen in Urine
Hemolysis
Hemorrhage in tissues
Causes of Reduced Urobilinogen in Urine
- Obstructive jaundice
- In biliary tract obstruction
- Reduction of intestinal bacterial flora
II. Chemical analysis Test for urobilinogen
Ehrlich’s aldehyde test
Ehrlich’s reagent (p-
dimethylaminobenzaldehyde) reacts with
urobilinogen in urine to produce a pink
color.
Take 5 ml of fresh urine in a test tube.
Add 0.5 ml of Ehrlich’s aldehyde reagent.
- Pink colour indicates normal amount of
urobilinogen.
- Dark red colour means increased amount
of urobilinogen.
II. Chemical analysis Test to differential between
urobilinogen and porphobilinogen
II. Chemical analysis Test for Urobilinogen
Reagent strip method:
This method is specific for urobilinogen.
Test area is impregnated with either p- dimethylaminobenzaldehyde
or 4-methoxybenzene diazonium tetrafluoroborate.
Principle
It is based on coupling reaction of bilirubin with diazonium
salt with which strip is coated.
Dip the strip in urine.
If it changes to blue colour then bilirubin is present.
II. Chemical analysis g) Blood
The presence of plenty intact red blood cells in urine is called as Hematuria
Causes
1. Disease of urinary tract

Glomerular disease: Glomerulonephritis, Berger’s Disease, Lupus Nephritis,

Henoch-s.Chonlein Purpura.
Non-glomerular disease: Calculus, Tumor, Infection, Tuberculosis,
Pyelonephritis, Hydronephrosis, Polycystic Kidney Disease, Trauma, After
strenuous physical exercise, Disease of prostate (Benign Hyperplasia or
carcinoma of prostate)
2.Hematological condition:

Coagulation disorders, sickle cell disease

II. Chemical analysis : Test for detection blood
1)Microscopic examination of urinary sediment:
Presences of 3 or more number of RBCs per HPF in 2 out of 3 collected samples are
define as Hematuria
2) Chemical tests:
Benzidine test:
Make saturated solution of benzidine in glacial acetic acid.
Mix 1ml of this solution with 1ml of hydrogen peroxide in a test tube
Add 2ml of urine
If green or blue color develops within 5 minutes ,the test is positive.
Orthotoludine test:
In this test instead of benzidine, orthotoluidine is used.
It is more sensitive than benzidine test.
Reagent strip test:
Various dip strips uses different chromogen (o-toluidine, tetramethylbenzidine).
II. Chemical analysis : Test for blood
Chemical tests are positive in Hematuria, Hemoglobinuria and
Myoglobinuria
II. Chemical analysis Causes of present of blood
II. Chemical analysisChemical 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 Ecoli, Salmonella, Proteus, Klebsiella) 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.
 URINALYSIS: Must and Must Not
Must
Dip the test areas of the strip in urine completely, but briefly, to avoid dissolving out
the reagents.
Read the test results carefully at the time specified in good light and with the test area
held near the specimen appropriate colour chart on the bottle label.
Protect the strips from moisture and excessive heat and light but do not refrigerate.
Replace the top on the storage container immediately after removing a strip.
Must Not
Do not use discoloured strips.
Do not touch the test areas.
Do not read the strips in direct sunlight.