Urine analysis – Chemical

examination -1
 Urine Examination
Routine urine analysis consists of three
important components

• Physical examination
• Chemical examination
• Microscopy
 Collection of urine specimen for
urine analysis
• The first voided morning sample is the best
for routine urine analysis
• Catheterised sample
• Sample obtained by supra public puncture
• 24 hrs urine collection for quantitative analysis
• Midstream, clean catch sample for
bacteriological analysis / culture
 Preservation of urine

• Urine is best examined within 4 hrs of collection.

• Refrigerated sample also gives good preservation for up to
24hrs
• In case of 24 hours urine analysis, preservatives like thymol
/ toluene / sodium azide / formalin can be added.
• Toluene - best all round preservative; it is added till a thin
layer is formed over the sample
• Formalin – One drop / 30 ml of urine; best for cast &
sediments
• Thymol – One crystal for a sample of urine.
• Con. Hcl – 10ml / 24 hrs urine, best for hormone estimation
 Physical Examination
• Volume
• Colour
• Odour
• Appearance
• Reaction / PH
• Specific gravity
 VOLUME
Normal volume- 1200 to 2000mL / day.

Polyuria >2000mL / 24 hr.

Oliguria <500mL / day.
Anuria - complete suppression of urine.

.
 Colour
Normal urine-Straw yellow (urochromes & urobilin)
a)Reddish brown- Increased urobilinogen or
porphyrins
b)Bright red-fresh blood.
c)Smoky brown-hemoglobin
d)Brownish yellow or green- Bile pigments
e)Milky white- Chyluria
f)Black – Alkaptonuria
g)Bright orange red – Rifampicin.
 Odour
• Normal fresh urine – aromatic odour
• Allowed to stand – ammoniacal smell

• Ketone bodies – fruity odour

• UTI – Foul smell

Normal pH- 4.6 to 8.0

 SPECIFIC GRAVITY

Solids in solutions in that urine

Urinometer is used for measuring specific
gravity of urine.

Normal specific gravity- 1.015 to 1.025

Fixed specific gravity
1.010-chronic glomerulonephritis
 Chemical examination
• Proteins
• Glucose
• Ketone bodies
• Bilirubin
• Bile salts
• Urobilinogen
• Blood
 Case scenario
• A 15years old boy complaining of puffiness
of face and passing smoky urine since five
days
• Record the physical characters of the given
urine sample.
• Find out the two abnormal chemical
constituents present in the urine.
Provisional diagnosis ??
• Acute Glomerulonephritis/ nephr

Chemical test
• Protein and Blood
 Chemical Examination

• Conventional methods.
• Reagent strips
 Reagent strip method
• Collect fresh urine specimen
in a clean dry container. Mix
well before testing.
• Remove one strip from bottle
and replace cap. Completely
immerse reagent areas to
the strip in urine and remove
immediately to avoid
dissolving out reagents.
 Procedure
• While removing, run the edge of the entire length
of the strip against the rim of the urine container
to remove excess urine.
• Hold the strip in a horizontal position to prevent
possible mixing of chemicals from adjacent
reagent areas and/or contaminating the hands
with urine.
• Compare reagent areas to corresponding colour
chart on the bottle label at the times specified.
• HOLD STRIP CLOSE TO COLOUR BLOCKS AND
MATCH CAREFULLY.
 Time limit for optimal results
• Glucose and bilirubin tests - 30 sec
• Ketone test - 40 sec
• Specific gravity test - 45 sec;
• pH, protein, urobilinogen and blood - 60 sec.
Colour changes after 2 minutes are of no
diagnostic value.
 Reagent strip - Principle
• Protein-error-of-indicator - The strip is
impregnated with Tetrabromophenol blue
buffered at pH 3.
• Various shade of green develops depending
on amount of protein present.
• This test is very sensitive to the presence of
albumin.
 Conventional method
Heat and acetic acid test:
• Fill 2/3rd of test tube with urine and heat top
column till it boils (top column alone is heated to
provide a comparison). The presence of protein is
indicated by white precipitate.
• Add 2-3 drops of 3% acetic acid to the above, if
cloudiness persists, it is due to proteins or else it
can be due to phosphates or carbonates. Proteins
can be graded as 1+ to 4+ depending on type of
precipitate.
• Trace – barely visible cloudiness against dark
back ground (<0.1 gm/dl)
• 1+ – definite cloudiness without granular
flocculation ((0.1 gm/dl)
• 2+ – heavy & granular flocculation (0.1 – 0.2
gms/dl)
• 3+ – Dense cloudiness with flocculation (0.2 –
0.4 gms/dl)
• 4+ – Curdy white precipitate (0.5 gms/dl)
 Other tests
• Sulphosalicylic acid test
• Esbach’s albuminometer- for quantitative
estimation of proteins
 Benzidine Test:
• Mix equal volume of benzidine and H2O2.
• Take 2-3 ml of urine in a test tube and add the
same volume of the above reagent to it.
• A blue colour indicates the presence of blood
or haemoglobin.
Principle-
• The peroxidase activity of hemoglobin
decomposes hydrogen peroxide releasing
nascent oxygen which in turn oxidizes
benzidine to give blue color
 Blood - Reagent strips
• The test is based on the peroxidase-like
activity of haemoglobin which catalyzes the
reaction of diisopropylbenzene
dihydroperoxide and tetramethylbenzidine
(or orthotoluidine).
• The resulting colour ranges from orange
through green to blue.
 Questions
• Define proteinuria and list the causes of
proteinuria
• Define microalbuminuria
• What is Bence-Jones protein? List the
conditions of Bence-Jones proteinuria.
 Questions
• Define haematuria.
• Define haemoglobinuria.
• Describe the method to differentiate them
• List the causes for haematuria and
haemoglobinuria
 Causes of hematuria
• Pre renal- bleeding diathesis,
hemoglobinopathies, malignant hypertension.
• Renal- trauma, calculi, acute & chronic
glomerulonephritis, renal TB, renal tumors
• Post renal – severe UTI, calculi, trauma,
tumors of urinary tract
Type Plasma color Urine color

Hematuria normal Smoky red

m/s-plenty of
RBC’s
Hemoglobunuria Pink, Red ,
hepatoglobin occasional
reduced RBC’s
Myoglobunuria Pink, normal Red, occasional
hepatoglobin RBC’s
 Urinalysis
Chemical examination Part 2
 Case history
• A 42 years old obese man complaining of increased
thirst and increasing frequency of passing urine
since two months
• Record the physical characters of the given urine
sample.
• Find out the two abnormal chemical constituents
present in the urine.
 Causes of symptoms
• Polyuria, polydipsia
- Deficiency of insulin reduces entry of glucose into muscle
and adipose tissue and glucose levels increase in blood.
- The hyperglycemia exceeds the renal threshold for
reabsorption and glycosuria occurs.
- Glycosuria induces an osmotic diuresis and thus polyuria
- Renal water loss and hyperosmolarity depletes intracellular
water, triggering the osmoreceptors of the thirst centers of
the brain causing intense thirst (polydipsia)
 2 abnormal substances
• Glucose
• Ketones
 Glucose
• Almost all the glucose that is filtered through the glomeruli is
reabsorbed by the proximal renal tubule
• Glycosuria represents an abnormal state.
• Two basic causes of glycosuria.
1. The level of blood glucose is so high that the renal tubules
are unable to reabsorb all that is presented. The level of
blood glucose at which it spills into the urine is called the
renal threshold – normally - 10 mmol/L (180 mg/dl)
2. Failure of the tubules to reabsorb all glucose at a level
where this should be possible -renal glycosuria.
 Methods of estimation
1. Benedict’s test
2. Dipstick method
 Benedict’s test
• Semi quantitative method.
Benedicts reagent
Mixture of sodium carbonate, sodium citrate and copper(II) sulphate
Principle
• The reducing sugar is oxidised by the cupric (Cu2+ ) complex of the
reagent which gets reduced to produces a cuprous (Cu+) complex
which precipitates. (Cupric ion is reduced by glucose (or other
reducing substances) to cuprous oxide). The colour of the precipitate
depends on the quantity of reducing substance present in the urine.
Procedure
• Take 5ml of Benedict’s solution in a test tube; add 8 drops of urine
(0.5ml). Heat to boiling for 2 minutes. Cool in water bath or running
tap water.
 Interpretation
• Negative – no colour
• Trace – Solution pale green
• 1+ - Definite cloudy green
• 2+ - Yellow – orange precipitate (1-0 gm/dl)
• 3+ - Orange, Red precipitate (1-2 gm/dl)
• 4+ - Brick red precipitate (>2.0 gm/dl)
• Why semi quantitative?
• Is it specific for glucose?
• Other substances that can give positivity in
Benedict’s test?
 Reagent strip method for glucose
• Specific for glucose
• Diastix®, Medi-Test® and Mission Glucose® are plastic strips
carrying glucose oxidase and a colour indicator like o-
toluidine or potassium iodide.
Principle
• It is based on a double sequential enzyme reaction.
• Glucose oxidase catalyses the formation of gluconic acid and
H2O2 from the oxidation of glucose.
• A second enzyme, peroxidase catalyses the reaction of H2O2
with a potassium iodide chromogen to oxidize the
chromogen to colours ranging from green to brown.
• 1) Catalysed by glucose oxidase
• Glucose + O2 → Gluconic acid + H2O2
• 2) Catalysed by peroxidase
• H2O2 + Chromogen → Oxidised chromogen (coloured) + H2O
Reagent strip
 Glycosuria
• Diabetes mellitus
• Thyrotoxicosis
• Acromegaly
• Cushing’s syndrome
• Renal glycosuria - presence of glucose in the urine in
association with normal or low blood glucose levels.
Asymptomatic. Due to mutations in the SLC5A2 gene coding
for the sodium-glucose co-transporter 2 (SGLT2) in the
proximal tubule.
Ketones
 Ketones
• Ketones or ketone bodies refers to three intermediate
products in the metabolism of fatty acids; acetone,
acetoacetic acid and beta-hydroxybutyric acid.
• Elevated concentrations of ketones are not generally found
in urine, as all these substances are completely metabolized,
producing energy, carbon dioxide and water.
 Mechanism of ketoacidosis
Insulin deficiency stimulates lipoprotein lipase
↓
Breakdown of adipose stores, increase in levels of FFAs
↓
Converted to ketones in liver
↓
Rate at which ketone bodies are formed may exceed the rate at
which acetoacetic acid and β-hydroxybutyric acid can be utilized
by peripheral tissues
↓
Ketonemia and ketonuria
↓
Dehydration will compromise urinary excretion of ketones
↓
Systemic metabolic ketoacidosis
 Methods
1. Rothera’s test
2. Dipstick or reagent strip method

Principle
Acetoacetic acid in an alkali medium reacts with the sodium
nitroprusside producing a magenta coloured complex

Na2[Fe(CN)5NO] + CH3COCH2COOH + 2Na(OH) → Na4[Fe(CN)5-

N=CHCOCH2COOH] (magenta) + H2O
Sodium nitroprusside + Acetoacetic acid + Alkali medium →
Pink-magenta complex + Water
 Rothera’s test
• Saturate 5ml of urine with ammonium sulphate in a test tube
and add 1 crystal of sodium nitroprusside and then trickle
down liquor ammonia along the sides of tube, so it layers on
the urine. A purple ring is formed at the junction of two
layers if ketone is present
 Reagent strip method
• The test is based on the nitroprusside reaction.
• The dipstick is coated with nitroprusside.
• A positive result is indicated by colour change to varying
shades of violet. It is specific only for acetoacetic acid.
 Ketones
• Detection of ketones
• Most methods detects acetoacetic acid
predominantly or acetoacetic acid and
acetone. ß-hydroxybutyric is seldom
detected.
 Ketonuria
• A positive test is associated with
- Diabetic ketoacidosis,
- Carbohydrate-free diets
- Starvation
- High fever
- Severe vomiting, diarrhoea.
• False trace results may be seen in highly coloured
urine and in patients taking levodopa.
Chemical examination of urine
Bile pigment and Bile salt
 Clinical history
• 34 year old man complaining of yellow
coloration of eye and itching all over the body
since six days
1. Record the physical characters of the given
sample.
2. Find out the 2 abnormal chemical
constituents present in the urine.
 RBC

Hemoglobin

Heme + globin

Heme = Iron + protoporphyrin

Protoporphyrin
Heme oxygenase
Biliverdin
Biliverdin reductase
Bilirubin
 Types of Bilirubin
• Direct (water soluble or conjugated bilirubin)
• Indirect (water insoluble or unconjugated
bilirubin)
 Types of jaundice
• Pre hepatic or hemolytic
• Hepatic
• Post hepatic or obstructive jaundice
 Liver function test
• Bilirubin
– total, direct and indirect
• Liver enzymes
– AST, ALT, GGT, ALP
• Protein
– Total, albumin, globulin and AG ratio
• Prothrombin time
 Bile constituents
• Bile pigment,bile salt, urobilin and
urobilinogen are excreted in urine
• Test for detection of bilirubin
– Fouchet’s test
– Urine strip method
 Fouchet’s test
– Add 2.5 ml of 10% barium chloride to 10 ml of
acidified urine
– Mix and filter
– Add one drop of Fouchet’s reagent to precipitate
– Green or blue color indicates presence of bilirubin
 Strip method for bilirubin
• Based on coupling of bilirubin with diazotized
2,4 - dichloroaniline in a strong acid medium
to form a brown purple azobilirubin
• Graded negative , 1+,2+,3+ depends upon
shading of color
 Bile salt
• Hay’s test
– When sulfur are sprinkled on surface of urine, if
bile salt are present , they sink to the bottom due
to property of bile salts to lower surface tension
 Urobilinogen in urine
• Ehrlich’s test
– Add 2.5 ml of 10% barium chloride to 10 ml of
acidified urine
– Mix and filter
– Add 0.5 ml of Ehrlich’s reagent to 2.3 ml of filtrate
– Allow it stand for 3 to 5 minutes
– Pink color ,viewed from top of the test tube
against a white background placed beneath the
bottom of test tube.
 Urine strip method for urobilinogen
• Modified Ehrlich’s test.
– P-diethylaminobenzaldehyde in conjugation with
color enhancer reacts with urobilinogen, in a
strongly acidic medium to produce pink red color
 Normal Pre hepatic Hepatic Post hepatic
Urine bilirubin negative Negative Positive or Positive
negative
Urine Normal Increased Increased Low or
urobilinogen absence
 Microscopic examination
• RBC
• Pus cells

• Crystal

• Cast
 Hyaline casts

Fever,Exercise,congestive heart failure,acute and

chronic glomerulonephritis.
 WBC CAST

Acute pyelonephritis,acute GN
 RBC CAST

Nephritic syndrome,Malignant HTN,SLE

GRANULAR & WAXY CAST

Chronic GN,Renal failure

 EPITHELIAL CAST

Toxic nephrosis,toxemia of pregnancy

 CRYSTALS

Calcium oxalate Triple phosphate

YEAST & TRICHOMONAS