Urine reflects the overall metabolic and kidney functions of the body. It can be used for diagnosis of structural and functional disorders of the urinary system as well as for monitoring diseases and detecting systemic disease processes. Urine can be analyzed for various properties including pH, protein, ketone bodies, blood, and bile salts and bile pigments to provide information about the patient's condition. Various tests exist for qualitative and quantitative analysis of these urine components.

1. Saikat Mitra

2.  Urine is the excretory waste product formed by
the kidney
 It reflects the overall metabolic and kidney
functions of the body
 Minimally invasive and cost-effective procedure
helpful in
 Diagnosis of structural and functional disorders of
urinary system
 Monitoring of diseases
 Detection of systemic disease process

4. Glomerular Filtration Tubular Reabsorption Tubular Secretions
GFR : rate at which plasma is
filtered by kidney glomeruli
• 125 ml of plasma /min or
180 Lit./day
Glucose, Amino Acid,Protein,
vitamin, Na, K, Ca, Mg,
water
Na, K, Cl
Na, Cl, Bicarbonate,
water
Urea, Uric Acid, Creat,
Urea
H+, K+, NH4+
1-2 Lit. Urine

5. Urine
Solid
Organic
Non-protein
Nitrogen
Organic
Acid
Sugars
Inorganic
Water

7. Clean catch midstream sample
• Specimen of choice (Routine + bacteriologic tests)
Catheterization of bladder
• in pts with difficult voiding
Suprapubic aspiration
• Infants & small children

8. Points to remember
• If a single specimen is submitted for multiple
measurements, bacteriologic examination should
be done first, provided that the urine has been
properly collected
• With paediatric patients and persons in acute renal
failure, only a small volume of urine may be available
for processing, and in such cases, a notation should
be made and the measurements most pertinent to
the diagnosis should be performed first

9.  Random sample sufficient
But
1st morning sample – specimen of choice (more conc.)
 For glycosuria – 2-3 hours after meals
 For urobilinogen – early afternoon sample
 For quantitative analysis – 24 hr sample
 Empty bladder & discard urine at 8 am
 Collect all urine for next 24 hrs including 8am sample
of next day
 Keep specimen in refrigerator during entire collection
period

10.  Ideal – fresh
 Refrigerate
 At room temperature
urea splitting bacteria
Urea NH3 + H+
NH4
+ pH
decomposition of casts
used by bacteria
Glucose in urine false –ve fo glycosuria
• Formation of crystals
• Loss of Ketone Bodies
• Oxidation of Bilirubin and Urobilinogen
• Disintegration of cellular elements

11. Preservatives Advantages Disadvantages Additional
Information
Thymol Preserves glucose and
sediments well
Interferes with acid
precipitation tests for
protein
Boric acid Does not interfere with
routine analyses other than
pH
Preserves protein and
formed elements well
May precipitate crystals
when used in large
amounts
Keeps pH at about 6.0.
Is bacteriostatic (not
bactericidal) at 18 g/L;
can use for culture
transport Interferes
with drug and hormone
analyses
Formalin
(formaldehyde)
Excellent sediment
preservative
Acts as a reducing agent,
interfering with chemical
tests for glucose, blood,
leukocyte esterase, and
copper reduction
Rinse specimen
container with formalin
to preserve cells and
casts
Toluene Does not interfere with
routine tests
Floats on surface of
specimens

13. Volume
Color
Odor
Appearance
Specific
gravity

14. Normal = 600-2000ml with night urine not in excess of
400 ml.
Polyuria- >3000ml/24 hrs
Oliguria- <400 ml/24 hrs
Anuria-complete cessation of urine
(>2 L/m2 in children)
<1 mL/kg/h in infants
<0.5 mL/kg/h in children

15.  Normal – pale yellow / straw
colored due to urochrome,
urobilin, uroerythrin
 Low specific gravity – pale
 High specific gravity – dark
 Acid urine darker than
alkaline

16. COLOR PATH CAUSE NON PATH CAUSE
Pale yellow to dark
amber
Normal
Yellow orange Bilirubin, urobilin Acriflavin, azogantricin, B
complex, pyridium,
nitrofurantoin, sulfa,
quinacrine, carrots
Milky white Pus, chyle Phosphate, carbonate
Pink – red Hb, Myoglobin, RBCs,
porphyrin,
porphobilirubin
Beets, Rifampicin, aminopyrin,
diphenyl hydantoin, M Dopa,
bromosulphthaline,
phenacetin, pyridium

17. COLOR PATH CAUSES NON PATH CAUSES
Red brown Porphobilinogen, uroporphyrin
Smoky Microscopic hematuria
Brown
black
Bilirubin, homogentisic acid
(alkaptonuria), melanin, indican,
Meth Hb, Myoglobin
Chloroquin, hydroquinone, iron
Rx, resorcinol, M Dopa, L
Dopa, nitrofurantoin,
Blue green Biliverdin, pseudomonas
infection
Acriflavin, amitryptiline, azure A,
methylene blue, B complex

18.  NORMAL – aromatic – organic acids
Odor Condition
Sweet/ fruity Ketones
Pungent Bacteria (NH3 production)
Maple syrup Maple syrup urine disease
Musty /mousy Phenylketonuria
Sweaty feet Isovaleric acidemia
Rancid butter/ fishy hypermethioninemia

19.  Turbidity evaluated by
holding the specimen in front
of a line of printed material
 Normal – clear
 Graded as
 clear
 slightly cloudy
 cloudy
 turbid

20. APPEARANCE CAUSES
Clear Normal
Cloudy Leukocytes, bacteria, epithelial cells,
amorphous phosphate (alkaline urine),
amorphous urates (acidic urine)
White ppt Amorphous phosphate
Amorphous pink ppt Amorphous urate
Hazy Mucus
Smoky Microscopic hematuria
Milky Fat, chyle

21. Ratio of weight of urine to weight of equal volume of pure
water
Urea (20%), sodium chloride (25%), sulphate, and phosphate
contribute most of the specific gravity of normal urine.
Normal range- 1.003 to 1.035

22. Measurement Methods
•Urinometer
•Refractometer
•Reagent strip
•Sp gravity beads
•Falling drop method

23.  Floating instrument calibrated at 200C
 Measures specific gravity at fixed temp (200C)
 Principle of buoyancy
 Urinometer floats higher in urine (denser than
water)
 Fill 15ml urine in cylinder (decant any turbid
material)
Put urinometer in jar by spinning
(do not let it touch side of cylinder)
Read lower meniscus
Correction
Actual = Observed – 0.003 for each
1g/dL protein
Actual = observed – 0.004 for each
1g/dL glucose
+/- 0.001 for every 30C temp rise or fall
respectively

24.  Measures refractive index of solution
 Scale transforms refractive index into
corresponding specific gravity value
 Refractive index =
velocity of light in air
velocity of light in solution
 Only one drop urine required (V/S
urinometer)

25.  Strip impregnated with pretreated poly
electrolyte susceptible to pKa changes in
relation to ionic conc which reads into sp gravity
 Ion dissociation
pH change
bromothymol blue indicator
color altered
 Spectrum of colors
Deep blue – green – yellow green

26. HYPERSTHENURIA
– high specific
gravity (>1.035)
• Dehydration
• Proteinuria
• Glycosuria
• Lipoid nephrosis
• False increase
(dextran,
radiographic dye)
HYPOSTHENURIA –
low sp gravity
(<1.007)
• Collagen diseases
• Pyelonephritis
• Protein
malnutrition
• Polydipsia
• DI
• Diuretics
- Coffee, alcohol
(natural diuretics)
ISOSTHENURIA –
fixed sp gravity of
1.010
• Total loss of
concentrating
power of kidney -
Poor tubular
absorption

28. pH
Reducing
substances
Protein
Ketone bodiesBlood
Bile salts and
bile pigments
Miscellaneous

29.  Rough assessment of pt’s acid base status &
ability of kidney to regulate acid base balance
 Normal range – 4.6 to 8.0
 Tested by-
 1.litmus paper
 2. pH paper
 3. Reagent strip method

30. Reagent Strip test
 Impregnated with methyl red & bromothymol
blue
 pH reflected by color change from orange (acid)
to green to blue (alkaline)
 Covers pH range 5 – 9
Litmus paper
 Blue Acidic Red
 Red Alkaline Blue

31. 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

32.  A small amount of protein (50 – 150 mg / 24
hrs) appears daily in the normal urine
 More than 150 mg/day is defined as
Proteinuria.

33. Protein % ofTotal 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

34. Qualitative tests
• Heat Precipitation test
• Sulphosalicylic acid test
• Heller’s method
• Reagent strip ( Albustix)
Quantitative tests
• Esbach’s Albuminometer
• Electrophoresis

35.  Principle – heat renders proteins insoluble & causes
them to coagulate.
Proteins are more susceptible to precipitation at pH
near their isoelectric point.
 Procedure – fill 2/3 test tube with urine
Hold bottom of test tube wit holder
Boil upper portion of tube for 2 min
Cloudiness
Protein/phosphate/carbonate
+ 3-5 drops 5-10% acetic acid + boil
Cloudiness disappears Cloudiness persists
phosphate / carbonates or increases - protein

36.  Detects albumin, globulin, mucoprotein, bence jones
protein
 Doesn’t detect Hb, myoglobin
 False +ve – tolbutamide, penicillin, radiographic dyes
 False –ve –highly alkaline urine of low sp gravity
 Interpretation
- No cloudiness
+/- Cloudiness barely present (~5 mg/dl)
+ Definite cloudiness but no granularity, no
flocculation (10-30 mg/dl)
++ Granular white ppt (40-100 mg/dl)
+++ Floccular ppt (200-500 mg/dl)
++++ Thick opaque ppt( >500 mg/dl)

37. Sulphosalicylic acid method
• Procedure
• Take 2 ml of clear urine in a
test tube.
• Add an equal volume of 30
% sulphosalicylic acid.
• Mix thoroughly, allow it to
stand for 10 minutes and
estimate the amount of
turbidity.
Nitric acid method
• Procedure
• Take 2 – 3 ml of
concentrated nitric acid in a
test tube.
• Carefully pour 5ml of clear
urine down the inner side
of the inclined test tube so
that the urine forms a layer
over the nitric acid.
• A ring of white precipitated
protein will form at the
interface. Estimates the
amount of precipitate.

38.  Principle – protein error of indicators i.e. point
of color change of some pH indicators is
different in presence of protein as compared to
their absence
 Indicator – tetrabromophenol blue
tetra chlorophenol tetra bromo
sulfophthalein
 Color change - Yellow green / blue
 Intensity of color ~ amount of protein
 False +ve – alkaline urine, phenazopyridine
 False –ve – dilute urine
 Very sensitive, detects 20mg% albumin

39. 24 hr sample
 Esbach’s albuminometer
Esbach’s reagent–
Picric acid- 5mg
Citric acid- 10mg
dissolved in 500 ml water
Procedure
urine up to mark “u”
+
esbach’s reagent up to mark “r”
Mix & stand for 24hrs
Read amount of protein ppt

40.  Definition : urinary excretion of albumin
▪ 20 to 200 ug/minute or
▪ 30 to 300 mg/24 hours
 Methods of detection :
 Albumin-creatinine ratio in random urine sample
 Measurement of albumin in early morning sample.
 Measurement of urine in 24 hr sample
 Reagent tablets or dipsticks
 Specific assays
▪ RIA
▪ ELISA
▪ Nephelometry

41.  Light chains in plasma cell dyscrasias
(multiple myeloma)
 Either kappa or lambda
 Tests done are:-
a) Heat precipitation test
b) Toluene sulfonic acid test
c) Electrophoresis – best
d) Bradshaw’s test
•Multiple myeloma
•Macroglobulinemia
•Lymphomas
•Amyloidosis

42. Heat 5ml of urine with a thermometer inside or in water
bath at 450 C
BJ proteins start precipitating at 600c
It dissolves if heated beyond & reappears when cooled
to 600 c
Albumin and glucose if +nt, ppt at 800c
filter
BJ come out in filtrate and reappear on cooling to 600 c

43. Method of Urine Concentration
•Dialysis against powdered sucrose
•MiliporeTechnique

44. Cu2+ Cu+
Sugars
• Glucose
• Fructose
• pentose
• galactose
Non sugars
• Ascorbic acid
• Uric acid, urates
• Glucuronides
• Salicylates
• Streptomycin
• Phenol, PAS

45. Various tests done are:-
 Benedict’s copper reduction test
 Fehling’s test
 Reagent strip ( Glucose – oxidase test )

46. Semi quantitative test
Detects the presence of reducing substance in
urine
Non specific test for reducing substances
Principle
cupric hydroxide
reducing subs
cuprous oxide (colored ppt)

47. Solution 1: Na citrate – 173 gms
Anhydrous Na Carbonate 100gms
Solution 2 Cu sulphate - 17.3 gms
Mix both solutions to make 1 litre in DW

48.  Procedure
5 ml of Benedict’s reagent in test tube
Boil
8 drops of urine
Boil for 5 minutes
note color of precipitate
FALSE POSITIVETEST
• Other sugars except sucrose
• Nonsugar subs - Creatine, Uric acid,
urates, Ascorbic Acid & Glucuronides
• Drugs- Salicylates, Cephalosporins,
Streptomycin

49. Fructose - Seliwanoff’s test
( Fructosuria- intake of grapes and citrus fruits)
Pentose - Bial’s test
Lactose - Methylamine test
( Lactosuria- late pregnancy, lactating women)
Glucose - Glucose oxidase test(Enzymatic method)

50.  DIASTIX / MULTISTIX / DIPSTIX contains
 Glucose oxidase
 Peroxidase
 Chromogen - O toluidine
potassium iodide
 Specific for glucose
 Strip dipped in urine for 10-30 seconds
 Color obtained is matched with a color chart
provided on the bottle
 Sensitivity - 0.1 mg%
False + Contamination with peroxide or
hypochlorite
False - Vitamin C if present inhibits enzymatic
reaction
- High Ketone levels

51.  Products of incomplete fat metabolism
 three discrete but metabolically related chemicals:
▪ acetoacetic acid (20%)
▪ β-hydroxybutyric acid (78%)
▪ Acetone (2%)

52. Tests done are:-
 Rothera’s test
 Gerhardt’s test
 Harts test
 Reagent strip / Ketostix / Ames test

53. Principle :
Acetoacetic acid/acetone + Sodium
nitroprusside (Na nitroferricyanide)
alkaline pH
ferropentocyanide (Purple color)
Rothera’s reagent
7.5 gm sodium nitroprusside
200 gm ammonium sulphate

54. Procedure :
4 ml of urine + few crystals of Na nitroprusside
Saturate with ammonium sulphate
+ few drops liquor NH3 along wall of tube
Wait for 2 min
Purple ring
 Interpretation-
no ring negative
Faint pink- purple ring traces (+)
Narrow dark purple ring (++)
Wide dark purple ring (+++)

55.  Specific forAcetoacetic acid
 10% ferric chloride
 5ml urine + 10% ferric chloride drop by drop
Port wine color

56.  Indirect test for β-Hydroxy butyric acid
20 ml urine + 20 ml DW +
few drops Acetic acid (removes acetone & AAA)
reduce vol to 10 ml
add 20 ml of DW
divide into 2 parts
+ 1ml H2O2 no H2O2 added
(converts βHBA into
acetone & AAA)
+ve Rothera’s reaction -ve Rothera’s reaction

57.  Based on Rothera’s test
Acetone /AAA+ Sod Nitroprusside
Alkali
Purple colored complex
a) Chemstrip: upto 10mg/dl AAA & 70 mg/dl
acetone
uses glycine
Beige Violet (60sec)
b) Multistix :up to 5-10 mg/dl AAA
Uses alkaline buffer
Pink Maroon (15 sec)

58.  Hematuria
 Hemoglobinuria
 Myoglobinuria
 Hemosiderinuria

59.  Benzidine reagent
1) Equal volume of saturated solution of
benzidine in glacial acetic acid
2) 3-6% H2O2
Mix 1ml of the two solutions
 Principle
H2O2 + peroxidase (in Hb) H2O+ O
Benzidine + O Blue Green color
(chromogen)
Glacial AA - lyse RBCs to release Hb

60.  Procedure
2ml urine + 2ml benzidine reagent
mix
Blue color
(blood/Hb)
 Interpretation
Faint green Traces
Green +
Greenish blue ++
Blue +++
Deep blue ++++

61. False + results
 Oxidising reagents like bleach & hypochlorite
 Microbial infection associated with UTI
False – results
 High specific gravity
 Protein & ascorbic acid
 Nitrite & formalin

62. Dipstick test / Heme tablet
Very small amount of Hb/RBCs detected
Principle
Org peroxide + peroxidase H20 + O
(reagent) (from Hb)
O + chromogen oxidised chromogen
(O-toluidine) (color)

64.  Na and K glycocholates and taurocholates
 Normally not present in urine
 Present in obstructive jaundice &
hepatocellular jaundice
 Decrease surface tension of urine – surfactant
action
 Test – Hay’s Sulphur test
20 ml of urine in a beaker
sprinkle sulphur powder
watch for 5 min
If it sinks to the bottom—Bile salts are present

65.  Normal excretion 2-4 mg/24 hrs (colorless)
 Causes of increased urobilinogen
 Hemolytic anemias
 Pre-icteric phase of infective hepatitis
 Causes of decreased or absent urobilinogen
 Obstructive Jaundice

66. Test for Urobilinogen :
EHRLICH’STEST
REAGENTS – Ehrlich Aldehyde reagent –
HCl 20ml
DW 80ml
P– dimethyl 2gm
aminobenzaldehyde
- Saturated Sod. Acetate solution
PRINCIPLE
 Ehrlich Reagent + aldehyde
urobilinogen Pink Colored
 ER + Porphobilinogen aldehyde
Pink complex

68.  confirmatory & D/D PBN
5 ml urine+5ml Ehrlich’s reagent+10ml sat Na acetate
sol
+ 5ml Chloroform, shake and mix
2 layers develop
upper clear aqueous upper colored aqueous &
& lower colored lower clear chloroform layer
chloroform layer
UBN +nt
+ 5ml butanol + shake
Pink lower pink upper butanol layer
Aqueous layer
PBN UBN

69.  Breakdown product of Hb.
 Causes - Obstructive Jaundice
Hepatocellular Jaundice
 Tests
 Fouchet’s test / Harrison spot test
 Foam test
 Icto test
 Strip test
 Smith iodine test
 Gmelin’s test

70.  Very sensitive (0.05 – 0.1 mg/dl )
 Fresh Sample
 Reagents
 10% Barium Chloride
 Fouchet’s reagent
▪ Trichloracetic acid 25 g
▪ DW 50ml
▪ 10% Ferric chloride 10ml
 Dilute H2SO4 or (NH4 )2 SO4 Solution

71. Principle
 BaCl2 + SO4 (in urine) BaSO4 ppt
 Bilirubin in urine adheres to ppt
 Bilirubin in ppt (yellow)
+FeCl3
+Trichloroacetic acid
Biliverdin (green)
 Bilicyanin - blue color

72.  Procedure
10ml urine + 3ml BaCl2 sol
mix & filter
ppt (bilirubin + Ba salt)
+ Fouchet’s reagent (few drops)
Green color
 Result
 No change in color – Negative
 Pale blue green color – Trace /+
 Darker blue green color – 2+ to 4+

73. ICTOTEST/TABLETTEST:
 Highly Sensitive (0.05 – 0.1mg/dl)
 Tablet containing diazo reagent, sulfosalicylic
acid, sodium bicarbonate & boric acid
Principle
 Bilirubin + Diazonium salts  Azobilirubin
(in Acidic Medium) (Blue / Purple)
REAGENTTEST STRIPS
Bilirubin + 2-4 dichloroaniline Cream buff to tan

74.  NITRITES
 E coli, klebsiella, proteus
 Nitrite + p-arsanilic acid (acid pH)
diazonium salt + benzoquinoline (PINK COLOR)
culture
 LEUKOCYTE ESTERASE
 Reagent-strip test for pyuria (leukocytes in urine)
 The intracellular esterases (bacterial) catalyze hydrolysis of
esters, releasing a product that reacts with a diazonium salt
to produce a colored product.

75. • Analyzes the intensity and color of light reflected
from the reagent areas of a urinalysis test strip.
• LEDs and (CMOS)
• Reads the color change in the urine strips after a
sample is applie
Wavelength Photometry
Refractive Index – for Sp. Gravity
•Semiautomated analyser
•Clinitek 200/200+/500
•Chemstrip Urine Analyzer
•Urisys 1800 System
•Fully automated analyser
•Clinitek Atlas
•Iris iChemVelocity

77.  A well mixed sample of urine (10 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

78.  Types of materials that may be found include:
 Red blood cells
 White blood cells
 Epithelial cells
 Casts
 Crystals
 Others

79.  TYPE
 Erythrocytes
 Leucocytes
▪ Neutrophils
▪ Eosinophils
▪ Lymphocytes
 Epithelial Cells
▪ Tubular cells
▪ Uroepithelial cells
▪ Squamous cells

80. MATRIX
• Hyaline
• Waxy
INCLUSIONS
• Granules-
proteins, cell
debris
• Fat globules-
triglycerides,
cholestrol
esters
• Hemosiderin
granules
• Melanin
granules- rare
PIGMENTS
• Haemoglobin
• Myoglobin
• Bilirubin
• Drugs
CELLS
• Erythocyte
casts
• Leucocytes :
neutrophils,
lymphocytes,
monocytes
• Renal tubular
epithelial cells
• Mixed cells :
erythrocytes,
neutrophils
and renal
tubular cells.
• Bacteria.

81. • Amorphous Urate
• Crystalline Urate
• Calcium Oxalate
Crystals found in normal acid urine
• Amorphous Phosphate
• Crystalline Phosphate
• Calcium Carbonate
• Ammonium Bi-urate
Crystals found in normal alkaline urine

82.  Tumor Cells
 Viral Inclusions
 Bacteria
 Fungi
 Parasites

83. Flow cytometry
• Sysmex UF-50, 100
Auto Particle Recognition (APR)
• Iris Q200
• A known volume of urine passes in front of a microscope objective.
•A camera takes 500 fields per sample.
•The particles are classified using a model of a neural network used by
the software: Auto-Particle Recognition (APR™).
•For samples with very abnormal particles, a user can visually confirm
the identification of those particles according to their morphological
details seen on the screen
•Minimum size of the recognized particles is 3 μm