URINE ANALYSIS

Types of Urine sample:

For routine examination a clean glass or plastic container can be used

1. First morning mid stream sample - ideal because

 Lowest pH which preserves the formed elements
 Larger quantity
 Concentrated

A mid –stream sample (patient is asked not to collect the initial part of
urine voided and collect only the middle part)

2. First morning mid stream, clean catch– Best for culture & sensitivity
and bacteriological studies
3. 24 hrs sample – For quantitative estimation of substances. Ex : Protein,
Sugar, Hormones. Collection should begin at 8 AM in the morning (discard
first voided sample) and continued till at 8 AM the next day

4. Post prandial sample (collected 2 hrs after meal) – Estimation of

glucose, urobilinogen
Other types in rare situation:
5. Catheterised sample – for bedridden patients and in obstruction of
urinary tract
6. Condoms – for collection in pediatric patients
7. Suprapubic aspiration

Ideally the urine should be tested as fresh as possible (within one hour) but
sometimes there may be a delay in transporting the urine to the laboratory.
To avoid any decomposition in such a case, preservatives are to be used.

Preservatives for Urine:

 Refrigeration at 4 degree C
 Thymol crystals
 Formalin
 Toluene– Overall best preservative
 Chloroform
 Dilute acids (sulphuric acid, hydrochloric acid, boric acid).

Urine is examined under three heading

1. Physical Examination
2. Chemical Examination
3. Microscopic Examination

Physical examination
 It consists of examination of volume, colour, appearance, odour,
specific gravity and pH of the provided sample.

It is determined by manual method, Urine Multistrip as well as by automated

analyser method.

1. Volume – Measured with measuring jar

Normal average volume = 1.5 litre/day (600-2500mL/day)
Increase in urine volume (>2500 mL/day) is called as POLYURIA, commonly
observed in:
a. Diabetes mellitus
b. Diabetes insipidus
c. Medication like diuretics
d. Excessive intake of water & cold weather
Decrease in urine volume (<500 mL/day) is known as OLIGURIA, commonly
observed in:
a. Acute glomerulonephritis
b. Acute Tubular Necrosis
c. Hypovolemia
d. Cardiogenic shock
e. Dehydration
f. Obstruction to urinary tract
Anuria - < 150ml/24hrs – seen in shock & renal failure.

2. Colour
Normal urine is pale yellow or straw coloured due to urochrome pigment.
Drug history should be asked
Abnormal colours
 Deep yellow – Jaundice
 Reddish/ pink - Pathological - hemoglobinuria, haematuria,
myogloinuria, porphyria ; Physiological - beetroot ingestion
 Smoky or smoky brown – blood along with protein/ albumin found in
acute nephritis.
 Orange – medication like rifampicin, concentrated urine.
 Milky – due to presence of chyle, fat, pus.
 Dark brown /black – Alkaptonuria (Homogentisic acid)

3. APPEARANCE :
 Normal - Clear
 Turbid – due to large number of pus cells called (pyuria) or due to large
amount of amorphous phosphate crystals (phosphaturia), urates,
bacteria, sperms, mucus substances, epithelial cells
 Milky – Chyle
 Foamy – Proteins, Bilirubin
4. Odour
 Normal odour of urine is mild aromatic due to presence of volatile
organic acids.
It may change to:
 Ammonical – if allowed to stand at room temperature for some time.
 Fruity – Presence of ketone bodies.
 Mousy – due to phenyl ketonuria.
 Fishy – Proteus infection/UTI
 Cabbage – Methionine malabsorption
 Rancid – Tyrosinemia

5. pH
Normal reaction of urine is acidic with pH 4.6 to 7.6.
Reaction is detected with the help of litmus paper, pH meter or pH paper
A. Litmus paper – ABR (Acid turns Blue litmus to RED)
B. pH paper: dip the paper in the urine , compare the colour change with
the control chart provided for each pH value.
C. pH meter with electrode – Digital method
D. Reagent Strip - The test is based on the double indicator principle
(Change in the color of the dye)

Causes of highly acidic urine

 Protein-rich diet (excessive meat)
 Urinary tract infection by E.coli
 Respiratory and metabolic acidosis – DM, fever, Starvation,
Dehydration
Causes of alkaline urine
 If specimen is allowed to stand at room temperature for some time it
becomes alkaline because of bacterial contamination or decomposition
(formation of ammonia)
 UTI with proteus infection

6. Specific Gravity
It is an estimate of the solute concentration of the urine (reflects
the density of the specimen)
It measures the concentrating ability of the kidney
Average normal specific gravity = 1.003 to 1.030
NaCl, Urea, sulphates & phosphates are responsible for normal specific
gravity of the urine.
A low and fixed value (1.010 - Isosthenuria) is found in chronic nephritis and
ADH deficiency
Specific gravity is low in (Hyposthenuria)
Diabetes insipidus and excessive water intake.
Specific gravity is high in
Concentrated urine as in dehydration, fever, albuminuria (nephrotic
syndrome), glycosuria (DM), adrenal insufficiency
Methods:
1.URINOMETER:
Urinometer is a hollow glass tube, closed at both the ends. It is thin at the
upper side and the lumen suddenly expands and is larger at lower end with a
heavy bulb at the bottom. The thin upper end shows graduations.
 Has calibrations from 1000 to 1060
 Readings are calibrated for the temperature of 20 degree Celsius
 Take an urinometer container ( wide mouthed, deep enough
cylinder or urine jar) and is filled up to 3/4 th of its volume with
urine.
 Remove any froth over the urine with blotting paper
 The urinometer should float freely without touching the walls of
the container.
 Note the lower meniscus reading
 Add or subtract 0.001 from the final reading for each 3 degree
above or below the calibration temperature (as specific gravity
decreases with rise in temperature)
2. Urine strips
Principle: The test is based on the principle of ion exchange,
which runs
between polyelectrolyte reagent and ions present in urine. Its result is colour
change of acid-base indicator bromothymol blue.

CHEMICAL EXAMINATION
Chemical analysis is done to detect presence of glucose, protein, ketone
bodies and blood and bile derivatives in urine.
1. Benedict's semiquantitative Test for glycosuria (Reducing
Substances)
 Done for detection of the presence of sugar in urine.
 Glucose is excreted in urine when the plasma glucose level is more
than renal threshold of 180mg/dl
 Reducing sugars encountered in urine are (positive benedict’s test)-
fructose, maltose, lactose except sucrose.
 Other non carbohydrate substances (give false positive benedict’s
test ) - ascorbic acid, salicylates, formaldehyde, homogentisic acid,
corticosteroids, certain antibiotics
Benedict's reagent is composed of cupric sulphate, sodium citrate
and sodium carbonate.
Principle: Reducing substances in urine reduces cupric ions in copper
sulphate (blue) to cuprous oxide in alkaline medium when heated.
The colour of which depends on the concentration of the sugar.
Procedure:
o Take 5 mL of Benedict's reagent in a test tube.
o Add 0.5 ml (8 drops) of urine.
o Heat it to boil for two minutes.
o Keep it for some time to cool.
 o Sediment settles down to bottom. Note the colour of precipitate.
o Depending on change of colour of precipitate the sugar content
is estimated as follows:
 Blue - negative
 Green – 0.5% (trace)
 Yellow – 1%
 Orange – 1.5%
 Red – 2%
 Brick Red - > 2%

Reagent strip (glucostix): Specific for glucose

o Based on glucose oxidase method (enzymatic reaction)
o This enzyme catalyses the oxidation of glucose by atmospheric oxygen
to form gluconic acid & H2O2 (first reaction)
o Second reaction – catalysed by peroxidase and converts H2O2 +
chromogen to oxidised chromogen + H2O
o This oxidised chromogen coloured compound that indicates the
glucose concentration.

Causes of Glycosuria
o Diabetes Mellitus
o Pregnancy (induced diabetes)
o Sepsis
o Renal glycosuria
o Drugs – OC pills, thiazides
o Alimentary glycosuria

2. Test for Proteins (proteinuria)

Normally up to 150mg/24hrs of protein is excreted in urine and composed of
proteins secreted by the tubules.
Causes of Proteinuria:
o Nephrotic syndrome (> 3.5gms/day)
o Chronic glomerulonephritis
o Nephrosclerosis
o UTI
o Chronic pyelonephritis
o Malignant Hypertension
o High fever
o Systemic lupus erythematosus
o Later stages of diabetes mellitus
o Orthostatic albuminuria (presence of albumin in urine of normal
individual after prolonged standing)
o Multiple myeloma (Bence jones protein)
1.Heat coagulation test: It is a qualitative test for the presence of protein
in urine
Principle:
It is based on the denaturation of the proteins by heat.
Procedure
o Take a small test tube and fill it up 3/4th level with urine. Small
tube is used so that a comparison column of urine is formed
between the lower and upper part of the test tube.
o Heat the upper 1/3rd of urine and use the lower 2/3rd as control.
o Watch for appearance of precipitate or cloudiness in the heated
part of urine.
o Add few drops of 10% acetic acid to the precipitate. If precipitate
persists it is because of proteins; if it dissolves in the acetic acid
then it is due to amorphous phosphates

Before heating check reaction of urine by litmus paper. It should be acidic in

reaction because proteins are best coagulated in acidic medium. If urine is
alkaline/neutral prior to test, acidify by adding few drops of 10% acetic acid.
Grading: The test tube is kept against a printed-paper and interpretation is
done.
No cloudiness – Negative
Traces – very fine turbidity, which can be observed only against direct light.
+ - Slight turbidity but print behind tube can be read.
++ - More turbidity, print is blurred to read.
+++- Print behind turbidity may not be read.
++++- Coagulum with most dense appearance. Print can't be seen and
coagulum may settle down to the bottom of the tube.

Other methods:
a. Sulphosalicylic Acid Test
 2-3 drops of a 20% Sulphosalicylic acid are added to 3 mL of urine.
 Urine becomes turbid if albumin is present.
 Proteins are denatured and gets precipitated after adding organic
acids.
b. Heller's Nitric Acid Test
 In a small test tube, 3mL of urine specimen is taken.
 By the side of the test tube pour 1 mL of conc.HNO3.
 Clear white ring appears at the junction if positive.
c. Reagent Strip Method
 Based on colour change of acid-base indicator, which is caused by
presence of proteins.
 It is particularly sensitive to albumin.
 Bromophenol coated readymade reagent strips are used.
 They are dipped in urine and change in colour is observed and read as
per colour code on bottle.
Bence Jones Proteins in urine
 These proteins appear on heating at temperatures between 30 to 40
degree C and disappear on further heating after 100 degree C. On
cooling the urine they again reappear.
 They are present in urine in cases of multiple myeloma
(immunoglobulin light chains)

3. Test for Ketonuria

Ketones excreted in urine are
1. Acetone
2. β hydroxy butyric acid
3. Acetoacetic acid

Ketonuria is caused by:

o Diabetic ketoacidosis
o Severe dehydration, fever
o Prolonged starvation
o Cachexia
o Hyperemesis gravidarum

Rothera's test is qualitative test for ketone bodies.

Principle: Acetone in urine after saturation with ammonium sulphate

crystals dissociate into acetate ions which combines with sodium
nitroprusside in alkaline medium to form permanganate coloured ring
(sodium nitroprusside acetate ion complex)

Procedure:
 Take 5 mL urine in a large test tube and saturate it with excess of
ammonium sulphate.
 Add few crystals of sodium nitroprusside.
 Slowly add 1-2 mL of liquour ammonia by the side of test tube.
 A purple ring is formed at the junction of two layers in case of ketone
bodies being present.
This test detects the presence of acetoacetic acid and acetones.

Other tests:
Gerhardt’s test
Reagent strip (ketostix)

4. Hay's Sulphur Test for Bile salts

Cholic acid and chenodeoxycholic acid conjugated with taurine & glycine are
the bile salts secreted in urine. The test is positive in
o Obstructive jaundice
o Cholestasis
Procedure
o Take 3 mL of urine in a small test tube and sprinkle sulphur powder on
it gently.
o If bile salts are present in urine then sulphur powder sinks to bottom of
the beaker.
o Take distilled water as a control – sulphur powder floats in it.
Principle:
Bile salts reduce surface tension of urine.

5.Test for Bile pigments:

Bile pigments includes bilirubin, biliverdin
Fouchet’s test:
Procedure:
o To 5ml of urine add 5ml of barium chloride, white precipitate forms.
Filter it and to the filtrate add fouchet’s reagent.
o Presence of bilirubin gives green colour to the filtrate.

Principle: Barium adsorbs bilirubin present in the urine and gives green
colour with fouchet’s reagent (Ferric chloride and trichloroacetic acid)

Gmelin’s test:
o Take 5ml of conc nitric acid in a test tube
o Slowly add 5 ml of urine along the sides of the test tube
o Look for a series of colours at the junction of acid and solution (Green,
blue, yellow or red)

Other tests:
Foam test
Smith’s test
Tablet test
Conjugated bilirubin is water soluble and can be excreted in urine while
unconjugated bilirubin is water insoluble.
So in obstructive jaundice conjugated bilirubin is excreted in urine.

Prehepatic Hepatic Post hepatic

(hemolytic) (obstructive)
Excessive red cell Dysfunction of hepatic Obstruction to biliary
breakdown overwhelms cells drainage.
liver’s ability to
conjugate bilirubin. Serum - Elevated
Serum - Increased Serum – elevated both conjugated bilirubin.
unconjugated bilirubin conjugated &
unconjugated bilirubin
in blood (mixed)
Hemolytic anemia Alcoholic liver disease Obstruction due to gall
Gilbert’s syndrome Viral hepatitis stones, strictures,
Criggler Najjar Medication tumours, abdominal
syndrome Autoimmune hepatitis mass lesions.

6.Benzidine Test for Occult blood

Procedure
o Prepare benzidine solution with glacial acetic acid (pinch of
benzidine powder + 1ml glacial acetic acid)
o Take 1 mL of benzidine solution in a test tube and then add 1 mL
of urine.
o Add 1ml of freshly prepared H2O2 solution by the sides of the
tube.
o Appearance of bluish green colour indicates the presence of
occult blood.

(Alternate for benzidine powder is orthotoludine)

Principle:
It is based on peroxidase activity of haemoglobin in RBC’s.
o H2O2 liberates oxygen in the presence of peroxidase
o Liberated oxygen oxidizes the chromogen (benzidine) to give coloured
complex.
o Oxidised benzidine gives bluish green colour

Occult blood in urine is found in case of:

Renal cause: acute glomerulonephritis, Polycystic kidney, strictures, stones,
tumours in the urinary tract.
Systemic cause: malignant hypertension, Bleeding diathesis, Transfusion
reaction, malaria.
Reagent Strip Method

MICROSCOPIC EXAMINATION OF URINARY SEDIMENTS

Procedure
o Morning first sample of urine is required for the microscopic
examination of urine.
o 10 mL urine is taken in test tube and then centrifuged at the
speed of 2000 RPM for 5minutes.
o The supernatant is discarded and a drop of sediment is taken on
a clean glass slide and a coverslip is placed over it.
There are two types of microscopic findings:
 Formed or organized sediments
 Unformed elements or unorganized sediments
Organized sediments:
 o Cells – pus cells (WBCs), RBCs, epithelial cells – reported as no/HPF
o Casts – Hyaline cast, epithelial casts, WBC casts, RBC casts, Lipid
casts, Waxy casts, broad casts – reported as no/LPF
o Others- bacteria, spermatozoa, fungi
Parasites – Trichomonas vaginalis, schistosoma ova
Artefacts like starch, fibers, hair etc., may also float in urine.
1. Pus cell (WBCs) – Round, small, granular cells measuring 10-12 μ in size
with multilobed nucleus. Reported as number of cells per high power field
(HPF) or exact cell count is done in Neubauer's chamber. In normal urine 0 -
4 cells/HPF can be seen.
Increased numbers of pus cells are seen in:
o Urinary tract infections (UTI)
o High-grade fever.
o Acute pyelonephritis.
o Prostatitis.
When large numbers of pus cells are seen in urine, it becomes turbid and is
called as pyuria.
Intermittent and sterile pyuria is seen in tuberculosis of kidney.
2. Red blood cells- RBCs are seen as round discoid structures. They are
translucent and 7-10 microns in diameter. RBC’s are crenated in
concentrated urine & swells up dilute urine.
Normal : 0-2 RBC / hpf
Presence of RBC in abnormal number is called as haematuria.
Increase numbers of RBCs are seen in:
a. Acute glomerulonephritis
b. Traumatic conditions
c. Stones, strictures, malignancies of urinary tract
f. BPH
g. Acute febrile illness
h. Severe exercise
3. Epithelial cells- most common element in normal urine. They are round
or polygonal cells many times larger than RBCs and pus cells. According to
the morphology, their origin in the genitourinary tract can be presumed.
Duke' s classified epithelial cells found in urine as.
a. Squamous cells:
b. Transitional cells:
c. Tubular epithelial cells: Seen in acute tubular necrosis, heavy metal
poisoning, graft rejection.

4.Casts - Casts are tubular or cylindrical structures with parallel borders and
rounded ends, found in urine. Abnormal number of casts in urine is known as
cylindriuria. Casts are reported as no of casts/LPF
If casts are seen in the urine, it indicates definitive renal pathology.
Casts always form in the tubules of kidneys. They are formed by the
precipitation of mucoproteins (Tamm Horsfall proteins).
Commonly seen casts are:
 Hyaline cast
 Granular cast
 Fatty cast
 Waxy cast
 Cellular casts – which include epithelial casts, pus cell casts
(leukocyte cast), RBCs cast and bacterial cast. These various cellular
elements are found trapped in the basic matrix. The causes of their
formation are different viz.:
 Epithelial Cast – Acute tubular necrosis, heavy metal poisoning,
renal transplant rejection.
 Pus cell (WBC) casts – acute pyleonephritis, acute
glomerulonephritis, lupus nephritis, nephritic syndrome, interstitial
nephritis.
 Red cell cast – Acute glomerulonephritis, lupus nephritis,
Goodpasture syndrome, renal infarction.

Unformed/ Unorganised Elements

They include crystals. Acidic and alkaline urine show different types of
crystals.