Urine analysis

Introduction
 Urine is formed in the kidneys, is a product
of ultrafiltration of plasma by the renal
glomeruli.

Collection of urine
 Early morning sample-qualitative
 Random sample- routine
 24hrs sample- quantitative
 Midstream sample-UTI
 Post prandial sample-D.M
 Catheterised- in infants, bedridden
patients
 Suprapubic needle aspiration

24 hour urine sample
1. For quantitative estimation of proteins
2. For estimation of vanillyl mandelic acid,
5-hydroxyindole acetic acid,
metanephrines.
3. For detection of hormones in urine
4. For detection of microalbuminuria

Specimen CollectionSpecimen Collection
Suprapubic Needle AspirationSuprapubic Needle Aspiration

Preservation of urine sample
 HCl- for 24 hr urinary sample preservation
for adrenaline, noradr, VMA, steroids.
 Toluene- as a physical barrier
 Boric acid- general preservative
 Thymol- inhibits bacteria, fungi
 Formalin- for preservation of formed
elements

 For routine analysis, preservatives should
be avoided as they interfere with reagent
strip technique and chemical test for
proteins.
 Sample should be examined within 1-2 hrs
of voiding.
 If delay is expected, sample can be kept in
refrigeration for max 8 hrs.

Urine examination
 Macroscopic examination
 Chemical examination
 Microscopic examination

Macroscopic examination
 Volume
 Color
 Odour
 Reaction or urinary pH
 Specific gravity
 Osmolality

Urinary volume
 Normal = 600-2000ml with night urine not in
excess of 400 ml.
 Polyuria- >2000ml/24 hrs
 Oliguria-<500ml/24 hrs
 Anuria-complete cessation of urine(<200ml)
 Nocturia-excretion of urine by a adult of >500ml
with a specific gravity of <1.018 at night
(characteristic of chronic glomerulonephritis)

Causes of polyuria
 Diabetes mellitus
 Diabetes insipidus
 Polycystic kidney
 Chronic renal failure
 Diuretics
 Intravenous saline/glucose

oliguria
 Dehydration-vomiting, diarrhoea,
excessive sweating
 Acute glomerulonephritis
 Congestive cardiac failure
Anuria
- Acute tubular necrosis
- Complete urinary tract obstruction

Color & appearance
 Normal= clear & pale yellow due to the
presence of various pigments called
urochrome.
1. Colourless- dilution, diabetes mellitus,
diabetes insipidus, diuretics
2. Milky-purulent genitourinary tract
infection, chyluria
3. Orange- urobilinogen
4. Red-beetroot ingestion,haematuria,
hemoglobinuria
5. Brown/ black- alkaptunuria, melanin

Urinary pH/ reaction
 Reaction reflects ability of kidney to
maintain normal hydrogen ion
concentration in plasma & ECF
 Normal= 4.6-8
 Tested by- 1.litmus paper
2. pH paper
3. Reagent strip method

Acidic urine
 Ketosis-diabetes, starvation, fever
 Systemic acidosis
 UTI by E.coli
 Acidification therapy
 High protein diet

Alkaline urine
 Strict vegetarian
 Systemic alkalosis
 UTI by pseudomonas or Proteus
 Alkalization therapy
 CRF

Odour
 Normal= aromatic due to the volatile fatty
acids
 Ammonical – bacterial action(E. coli)
 Fruity- ketonuria, starvation
 Musty- Phenylketonuria
 Fishy- UTI with Proteus
 Rancid- Tyrosinemia

Specific gravity
 Depends on the concentration of various
solutes in the urine.
 Normal range- 1.003 to 1.035
 Measured by-urinometer
- refractometer
- reagent strip method
- falling drop method

Urinometer
 This method is based on the principle of
buoyancy.
 Take 2/3 of urinometer container with urine
 Allow the urinometer to float into the urine
 Read the graduation at the lowest level of
urinary meniscus
 Correction of temperature & albumin is a
must.
 Urinometer is calibrated at 15or 200
c
So for every 3o
c increase/decrease add/subtract
0.001

High specific
gravity(hyperosthenuria)
 Causes
All causes of oliguria
Gycosuria, DM, Dehydration, nephrotic
syndrome.

Low specific
gravity(hyposthenuria)
 All causes of polyuria except gycosuria
 DI, pyelonephritis, glomerulonephritis.
 Fixed specific gravity (isosthenuria)=1.010
Seen in chronic renal disease when kidney
has lost the ability to concentrate or dilute

Osmolality
 Normal adult with normal fluid intake wil
produce urine of 500-850 mOsm/kg water.
 The normal kidney is able to produce
urine osmolality in the range of 800-1400
mOsm/kg water in dehydration and
minimal osmolality of 40-80 mOsm/kg
water during diuresis.

Chemical examination
 Proteins
 Sugars
 Ketone bodies
 Bilirubin
 Bile salts
 Urobilinogen
 Blood

Tests for proteins
 Test – HEAT & ACETIC ACID TEST
 Principle-proteins are denatured & coagulated
on heating to give white cloud precipitate.
 Method-take 2/3 of test tube with urine, heat only
the upper part keeping lower part as control.
 Presence of phosphates, carbonates, proteins
gives a white cloud formation. Add 1-2 drops of
10% acetic acid, if the cloud persists it indicates
it is protein(acetic acid dissolves the
carbonates/phosphates)

Other Tests for Protien
 Nitric acid test
 Sulphosalicylic acid test
 Test with Esbach’s reagent
 Protienreagent strip
 Biuret method

Protein % of Total Daily Maximum
Albumin 40% 60 mg
Tamm-Horsfall 40% 60 mg
Immunoglobulins 12% 24 mg
Secretory IgA 3% 6 mg
Other 5% 10 mg
TOTAL 100% 150 mg
Proteins in “Normal” UrineProteins in “Normal” Urine

Causes of proteinuria
 Glomerular proteinuria: due to increased
permeability of glomerular capillary wall.
Selective( only albumin n transferrin bands seen)
Nonselective: pattern same as serum
e.g. nephrotic syndrome
 Tubular proteinuria: in acute n chronic
pyelonephritis, heavy metal poisoning, TB
kidney etc.

 Overflow proteinuria: Bence jones
proteins(plasma cell dyscrasia),
hemglobin( intravascular hemlysis),
myoglobin(skeletal muscle trauma)
 Hemodynamic proteinuria: seen in high
fever, hypertension, heavy exercise, CCF
etc.
 Post-renal proteinuria: caused by
imflammatory or neoplastic conditions in
renal pelvis, ureter, bladder, prostate or
urethra.

microalbuminuria
 It is presence of albumin in urine above
normal level bt below detectable range of
conventional urine dipstick method.
 Defined as urinary excretion of 30 to 300
mg/24 hrs of albumin in urine.

Significance of microalbuminuria
 an indicator of subclinical cardiovascular
disease
 an important prognostic marker for kidney
disease
 in diabetes mellitus (earliest sign of renal
damage in DM)
 in hypertension
 increasing microalbuminuria during the first 48
hours after admission to an intensive care unit
predicts elevated risk for acute respiratory failure
, multiple organ failure , and overall mortality

Detection of microalbuminuria:
 Methods for detection :
 Measurement of albumin creatinine ratio
in random urine sample.
 Measurement of albumin in early morning
sample.
 Measurement of urine in 24 hr sample.

Bence Jones proteins
 These are monoclonal immunoglobulin light
chains (kappa or lamda) synthesized by
neoplastic plasma cells.
 seen in multiple myeloma, macroglobulinemias,
primary amyloidosis.
 Test- Thermal method(waterbath):
Proteins have unusual property of
precipitating at 400
-600
c & then dissolving
when the urine is brought to boiling(1000
c) &
reappears when the urine is cooled.

Test for sugar Test-BENEDICT’S TEST(semiquantitative)
 Principle-benedict’s reagent contains
cuso4.In the presence of reducing sugars
cupric ions are converted to cuprous oxide
which is hastened by heating, to give the
color.
 Method- take 5ml of benedict’s reagent in a
test tube, add 8drops of urine. Boil the
mixture.
 Blue= negative
 Yellow=+(<0.5%)
 green=++(0.5-1%)
 Yellow-orange=+++(1-2%)
 Brick red=++++(>2%)

Benedict’s test
 Detects all reducing substances like
glucose, fructose, & other reducing
sustances.
 Sensitivity of the test is about 200 mg
reducing substance per dl of urine.
 To confirm it is glucose, dipsticks can be
used (glucose oxidase)

Reagent strip method:
 Specific for glucose.
 Based on glucose oxidase peroxidase
reaction.
 More sensitive (sensitivity- 100 mg
glucose/dl)
 Glu + oxygen---- gluconic
acid+hydrogen peroxide
 Hydrogen peroxide+chromogen--
oxidised chromogen(blue)+ H2O

Glycosuria
 Other Methods of detecting glycosuria
 Fehling’smethod
 Osazone test

Causes of glycosuria
 Glycosuria with hyperglycaemia-
diabetes,acromegaly, cushing’s disease,
hyperthyroidism, drugs like corticosteroids.
 Glycosuria without hyperglycaemia-
renal tubular dysfunction

Ketone bodies
 3 types
 Acetone
 Acetoacetic acid
 β-hydroxy butyric acid
 They are products of fat metabolism

Rothera’s test
 Principle-acetone & acetoacetic acid react
with sodium nitroprusside in the presence
of alkali to produce purple colour.
 Method- take 5ml of urine in a test tube &
saturate it with ammonium sulphate. Then
add one crystal of sodium nitroprusside.
Then slowly run the liquor ammonia along
the sides of the test tube.
 Formation of purple coloured ring at
junction indicates + test

Other tests:
 Acetest tablet test
 Ferric chloride test(Gerhardt’s test)
 Reagent strip method
 Hart’s test for beta hydroxy butyric acid

Causes of ketonuria
 Diabetes
 Non-diabetic causes- high fever,
starvation, severe vomiting/diarrhoea
 Glycogen storage diseases

Bilirubin
 Test- fouchet’s test.
In 5 ml of urine add 2.5 ml of 10% Barium
chloride and mix well. Then filter to obtain
precipitate. To ppt add 1 drop of fouchet’s
reagent. Development of blue green colour
indicate + test.
Principle: Bilirubin Adsorbs to the Barium Chloride
and results in green color formation when
fouchet’s reagent is added.
 Causes
 Liver diseases-injury,hepatitis
 Obstruction to biliary tract

Other tests:
 Foam test
 Gmelin’s test (nitric acid)
 Lugol’s iodine test
 Reagent strip with diazo reagent

Urobilinogen
 Test- ehrlich test
In 5 ml of urine add 0.5 ml of Ehrlich’s
reagent(HCl 20 ml, d/w 80 ml, p-
dimethylaminobenzaldehyde 2 gm). Allow
to stand for 5 min. development of pink
colour indicates +test.
 Causes-hemolytic jaundice, early
hepatitis, hepatocellular jaundice.

Bile salt:
 Hay’s sulphur test:
In 5 ml of urine sprinkle a pinch of
sulphur particles. If bile salt is present
sulphur particles will sink to the bottom
because bile salts lowers the surface
tension of urine.

Blood in urine
 Test- BENZIDINE TEST
 Principle-The peroxidase activity of hemoglobin
decomposes hydrogen peroxide releasing
nascent oxygen which in turn oxidizes benzidine
to give blue color.
 Method- mix 2ml of benzidine solution with 2ml
of hydrogen peroxide in a test tube. Take 2ml of
urine & add 2ml of above mixture. A blue or
green color within 5 min indicates + reaction.

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,hepatoglob
in reduced
Red ,
occasional
RBC’s
Myoglobunuria Pink, normal
hepatoglobin
Red, occasional
RBC’s

Microscopic examination
 A well mixed sample of urine (12 ml) is
centrifuged in machine for 5 min at 1500 rpm.
The top liquid part (the supernatant) is
discarded. A drop of urine left at the bottom of
the test tube (the urine sediment) is placed on
glass slide and covered with cover slip. It is
examined under high power.

Contents of normal urine m/s
 Contains few epithelial cells, occasional
RBC’s, few crystals.

EPITHELIAL CELLS.
squamous cell Transitional cell Tubular
epithelial cell

Crystals in urine
Crystals in acidic
urine
 Uric acid
 Calcium oxalate
 Cystine
 Leucine
Crystals in alkaline
urine
 Ammonium
magnesium
phosphates(triple
phosphate crystals)
 Calcium carbonate

Microscopic ExaminationMicroscopic Examination
Calcium Oxalate CrystalsCalcium Oxalate Crystals

Calcium Oxalate CrystalsCalcium Oxalate Crystals
Dumbbell ShapeDumbbell Shape

Triple Phosphate CrystalsTriple Phosphate Crystals

Urate CrystalsUrate Crystals

Leucine CrystalsLeucine Crystals

Cystine CrystalsCystine Crystals

Ammonium Biurate CrystalsAmmonium Biurate Crystals

Cholesterol CrystalsCholesterol Crystals

casts
 Urinary casts are cylindrical aggregations
of particles that form in the distal nephron,
dislodge, and pass into the urine. In
urinalysis they indicate kidney disease.
They form via precipitation of
Tamm-Horsfall mucoprotein which is
secreted by renal tubule cells.

Types of casts
 Acellular casts
Hyaline casts
Granular casts
Waxy casts
Fatty casts
Pigment casts
Crystal casts
 Cellular casts
Red cell casts
White cell casts
Epithelial cell cast

Hyaline casts
 The most common type of cast, hyaline
casts are solidified Tamm-Horsfall
mucoprotein secreted from the tubular
epithelial cells
 Seen in fever, strenuous exercise,
damage to the glomerular capillary

Hyaline CastHyaline Cast

Granular casts
 Granular casts can result either from the
breakdown of cellular casts or the
inclusion of aggregates of plasma proteins
(e.g., albumin) or immunoglobulin light
chains
 indicative of chronic renal disease

Granular CastGranular Cast

Waxy casts
 waxy casts suggest severe,
longstanding kidney disease such as
renal failure(end stage renal disease).

Waxy CastWaxy Cast

Waxy casts

On fluoroscent microscopy

Fatty casts
 Formed by the breakdown of lipid-rich
epithelial cells, these are hyaline casts
with fat globule inclusions
They can be present in various disorders,
including
 nephrotic syndrome,
 diabetic or lupus nephropathy,
 Acute tubular necrosis

Fatty CastFatty Cast

Pigment casts
 Formed by the adhesion of metabolic
breakdown products or drug pigments
 Pigments include those produced
endogenously, such as
 hemoglobin in hemolytic anemia,
 myoglobin in rhabdomyolysis, and
 bilirubin in liver disease.

Crystal casts
 Though crystallized urinary solutes, such
as oxalates, urates, or sulfonamides, may
become enmeshed within a hyaline cast
during its formation.
 The clinical significance of this occurrence
is not felt to be great.

Red 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.

RBCs CastRBCs Cast

White blood cell casts
 Indicative of inflammation or infection,
 pyelonephritis
 acute allergic interstitial nephritis,
 nephrotic syndrome, or
 post-streptococcal acute
glomerulonephritis

WBCs CastWBCs Cast

Epithelial casts
 This cast is formed by inclusion or
adhesion of desquamated epithelial cells
of the tubule lining.
These can be seen in
 acute tubular necrosis and
 toxic ingestion, such as from mercury,
diethylene glycol, or salicylate.

Tubular Epith. CastTubular Epith. Cast

Miscellaneous structures in
urine:

Urine dipsticks
 Urine dipstick is a narrow plastic strip which
has several squares of different colors
attached to it. Each small square represents
a component of the test used to interpret
urinalysis. The entire strip is dipped in the
urine sample and color changes in each
square are noted. The color change takes
place within 2 minutes from dipping the
strip. If read too early or too long after the
strip is dipped, the results may not be
accurate.

Chemical AnalysisChemical Analysis
Urine DipstickUrine Dipstick
Glucose
Bilirubin
Ketones
Specific Gravity
Blood
pH
Protein
Urobilinogen
Nitrite
Leukocyte Esterase

 The squares on the dipstick represent the
following components in the urine:
∀ specific gravity (concentration of urine),
∀ acidity of the urine (pH),
∀ protein in the urine (mainly albumin),
∀ glucose (sugar),
∀ ketones
∀ blood
∀ bilirubin and
 Urobilinogen
 Nitrites
 Leukocyte esterase

 The main advantage of dipsticks is that
they are
1. convenient,
2. easy to interpret,
3. and cost-effective

The main disadvantage is that the
1. Information may not be very accurate as
the test is time-sensitive.
2. It also provides limited information about
the urine as it is qualitative test and not a
quantitative test (for example, it does not
give a precise measure of the quantity of
abnormality).

Automated urine analyser:
 It is based on the principle of flow
cytometry.
 This system classifies particles based on
fluoroscent intensity ( cells are stained
with 2 fluoroscent dyes), electrical
impedance and forward angle light scatter.
 Next generation automated image based
urinalysis system iQ200 is a walk away
system.

 This system uses flow imaging analysis
technology and Auto Particle Recognition
software.
 It requires minimum of 3 ml of urine and
quantitates particles in 2 microlitre of
sample.
 Can analyse 60 urine samples per hr.

References:
 Henry’s Clinical diagnosis and
Management of Laboratory Methods
 Clinical Pathology Kawthalkar
 Clinical Pathology Sabitri Sanyal
 Basics of Body fluids by Akhil Bansal
 Internet

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