Urine examination

1. URINE EXAMINATION
DR. SAMEERA MEHTA
DR. MADHULIKA KATEY

2. INDICATIONS OF URINE ANALYSIS
1. Suspected renal diseases
 Glomerulonephritis
 Nephrotic Syndrome
 Pyelonephritis
 Renal failure
2. Diagnosis of Urinary tract infection
3. Metabolic disorders i.e. Diabetes mellitus
4. Differential diagnosis of jaundice
5. Detection of Plasma celldyscrasias
6. Diagnosis of pregnancy

3. Collection of urine: Time of collection
1. Single Specimen :
 First morning voiding:
 Most concentrated
 Acidic
Used for :
 Routine examination
 Fasting Glucose
 Protein
 Nitrite
 Pregnancy test
 Microscopic analysis of cellular
elements
 Orthostatic proteinuria
 Bacteriological analysis

4. Collection of urine: Time of collection
The Random Specimen:
• Can be collected at any point of time
• Routine examination
Post-prandial specimen:
• Collected two hours after a meal in the afternoon
• Insulin therapy monitoring in DiabetesMellitus
• Urobilinogen

5. Collection of urine: Time of Collection
2. 24 hr Specimen:
First urine is discarded inmorning
Clean 2 litres bottle with cap isused
Whole day and night urine collected and next day first urine
in morning is also collected
Preserved at 4-6◦C duringcollection
After collection immediate transportation to lab
Thoroughly mixed and aliquot used for testing.

6. Collection of urine: Time of collection
• 24- hr Specimen: used for quantitative estimation of
• Protein
• Hormones (catecholamines, cortisol, etc)
• Creatinine
• Electrolytes

7. Collection method
Container used should be:
 Clean
 Dry
 Leakproof
 Wide mouth
 Wide flat bottom
 Disposable
 Made of clear material
 50 ml capacity

8. Collection methods
1. Midstreamspecimen:
o Used for all types ofexamination
o Collected after voiding initial half of urine
2. Clean-catchspecimen:
o Used for bacteriologicalculture
o Urethral opening- cleaned with soap and water
o Collected after voiding initial half of urine
3. Catheterspecimen:
o Used for bacteriologicalculture
o For bed ridden and obstructed urinary tract patient
4. Infants:
o Aspiration done above the symphysis pubis
o Plastic bag specimen can also be used

9. Collection methods
4. Three glass specimen :
o Used for detection of prostate infection
o UTI- pus cells and bacteria are present in 2nd container
o Prostate infection- pus cells and bacteria are more in 3rd
container

10. Changes which Occur in Standing Urine
at Room Temperature
 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.

11. Changes which occur in standing urine at Room
Temperature:
 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

12. PRESERVATION OF URINE SAMPLE
Ideally urine must be tested within TWO hours after collection.
Refrigeration (4-6°C):
 Preserves urine sample up to 8 hours max 24 hrs
 S/E -Raises specific gravity
Ppts phosphates.
 Used for routine urine analysis
In 24 hour urine sample :
1. Toluene
2. Boric acid
3. Thymol
4. Formalin
5. HCl

13.  Proper labelling
 Sterile container and
appropriate
preservative
 Transportation delays
 Signs of contamination

14. Physical examination of urine
• Volume
• Color
• Odor
• Appearance
• Specific gravity

15. Volume
24 hrs. average urine output- 600 to 2000 ml (adult)
POLYURIA
>2000 ML/24 hrs
• Diabetes Mellitus
• Diabetes Insipidus
• Chronic renal
failure
• Diuretic therapy
OLIGURIA
<400 ML/24 hrs
• Febrile states
• Acute
glomerulonephritis
• Congestive cardiac
failure
• Dehydration
ANURIA
<100ML/24 hrs
• Acute tubular
necrosis
• acute
glomerulonephritis
• complete urinary
tract obstruction

16. COLOR
COLOR CONDITION CAUSE
Yellow color Normally
Urochrome
Urobilin
Uroerythrin
Red 1. Menstrual flow
2. Hematuria
3. Drugs and dyes
4. Myoglobin
5. Porphyria
RBCS
RBCS
Rifampicin, deferoxamine.
Muscle injury
Deranged heme

17. COLOR
COLOR CONDITION CAUSE
Yellow brown / greenish brown Jaundice (yellow foam on
shaking)
Concentrated urine (white
foam on shaking)
Bilirubin
Decreased fluid
Yellow red / orange red Hemolysis Urobilin urobilinogen
Dark brown / black urine Hemoglobinuria
Drugs
Methemoglobin
Levodopa
Blue-green urine Infections
Drugs
Pseudomonas
Hartnups disease
Amitriptyline
Cimetidine
Promethazine.

18. ODOUR
Normal
faint, aromatic odor
Cystinuria Rotten eggs
Hawkinsinuria
Swimming pool
Ketoacidosis
Sweet, fruity
Isovaleric acidaemia and glutaric acidaemia
Sweaty feet
Maple syrup urine disease (MSUD)
Maple syrup
Methionine malabsorption
Cabbage, hops
Phenylketonuria Mousy, musty
Trimethylaminuria Rotting fish

19. Appearance
Normal, freshly voided urine is clear in appearance
Cause Appearance Diagnosis
Amorphous phosphates White and cloudy on
standing in alkaline urine
Disappear on addition of
a drop of dilute acetic
acid
Amorphous urates Pink and cloudy in acid
urine
Dissolve on warming
Pus cells Varying grades of
turbidity
Microscopy
Bacteria Uniformly cloudy; do not
settle at the bottom
following centrifugation
Microscopy, Nitrite test

20. Specific Gravity (SG)
 Normal range :1.003 to 1.030
 Urea (20%), sodium chloride (25%), sulfate, and
phosphate
 SG increases as solute concentration increases
 SG decreases when temperature rises

21. Specific Gravity (SG)
Hyposthenuria
diabetes insipidus
pyelonephritis
diuretics
alcohol
Hypersthenuria
Diabetes mellitus
Glycosuria
Albuminuria
Fever
Dehydration
Isosthenuria - Low and fixed SG at 1.010 due to loss of
concentrating ability of tubules seen in end stage renal failure.

22. Methods for measuring SG
Urinometer method
Refractometer method
Reagent strip method
Falling drop method.

23. Urinometer method
 Based on the principle of buoyancy
 Fill urinometer vessel with 3/4th urine
 Insert urinometer –SPINNING MOTION
 Read the bottom of meniscus
 Take necessary precautions and make
necessary corrections
Correction for temperature : add /sub 0.001 for every 3 degree rise / fall
Correction for glucose : subtract 0.004 per mg/dl
Correction for protein : subtract 0.003 per mg/dl

24. Refractometer method
 Principle - Refractive index of solution
directly proportional to dissolved solids in the
solution
 Refractometer determines the refractive index
of total soluble solids in urine.
 Higher the concentration of soluble solids in
urine higher refractive index so higher SG
 The method is simple and requires only 1-2
drops of urine.
 Result is read from a scale or from digital
display.

25. Reagent strip method
 Indirect method.
 concentration of ions in urine correlates with
SG.
 Depending on the ionic strength of urine, a
polyelectrolyte will ionize releasing hydrogen
ions in proportion.
 This causes a change in color of ph indicator
(bromothymol blue) and this is translated to
specific gravity values.

26. Falling drop method.
 Direct method
 more accurate than the refractometer and more precise than the
urinometer.
 Utilizes a specially designed column filled with water-immiscible
oil.
 A measured drop of urine is introduced into the column, and as
this drop falls, it encounters two beams of light; breaking the first
beam starts a timer, and breaking the second turns it off.
 The falling time is measured electronically and is expressed as a
specific gravity.

27. CHEMICALEXAMINATION
The chemical examination is carried out for the
following substances :-
 pH
Proteins
Glucose
Ketones
Bilirubin
Bile salts
Urobilinogen
Blood
Hemoglobin
Myoglobin
Nitrite or leukocyte esterase

28. Reaction and pH
 pH is the scale for measuring acidity or
alkalinity.
 Normal value ranges from 4.6-8
 Various methods for determination of reaction of
urine:
 Litmus paper
 pH indicator paper
 pH meter
 Reagent strip tests

29. Reaction and pH
 1. Litmus paper test:
 A small strip of litmus paper is dipped in urine and any
color change is noted.
 2. pH indicator paper:
 Indicators -bromothymol blue and methyl red
 Color change is noted
 Range - 5 to 9 ( orange, green, blue)

30. Reaction and pH
 3. pH meter:
 An electrode of pH meter is dipped in urine sample and pH
is read off directly from the digital display.
 Gives accurate results
 4. Reagent strip test:
 contains polyionic polymer bound to H+
 On reaction with cations in urine, H+ is released causing
change in color of the pH-sensitive dye.

31. Reaction and pH
Acidic Urine:
 Ketosis
 diabetes mellitus
 Starvation
 Fever
 Urinary tract infection
caused by –
Escherichia coli
 High protein diet.
Alkaline Urine:
 Severe vomiting
 Old ammoniacal urine sample
 Chronic renal failure
 Urinary tract infection caused by
bacteria that split urea to
ammonia - Proteus or
Pseudomonas
 Vegetarian diet

32. CHEMICALEXAMINATION
Proteins
 Normal -150 mg/24 hours
 These include:
 proteins from plasma (albumin)
 proteins derived from urinary tract
 Tamm-Horsfall protein-*normal mucoprotein
secreted by ascending limb of the loop of
Henle.
 Secretory IgA
 proteins from tubular epithelial cells,
leucocytes and other desquamated cells.

33. CHEMICALEXAMINATION
Proteins
 Proteinuria refers to protein excretion in urine >150 mg/24
hours in adults.
 Causes:
 1. Overflow proteinuria (Pre-renal proteinuria): Proteinuria
associated withincreased production of low molecular weightproteins,
 Bence jones protein (plasma celldyscrasias)
 Hemoglobin (intravascular haemolysis)
 Myoglobin (skeletal muscle trauma)
 Lysozyme (acute myeloid leukemia type M4 orM5)

34.  2. Renal proteinuria- 2 main types –glomerular and tubular.
 Glomerular proteinuria: Proteinuria due to increased permeability of
glomerular capillary wall
Selective Nonselective
• When glomeruli can retain larger
molecular weight proteins but allow
passage of comparatively lower molecular
weight proteins like albumin and
transferrin.
• This is generally seen in early stages.
• In electrophoresis, albumin and transferrin
bands are seen.
• With further glomerular damage larger
proteins (γ globulins ) are also excreted
along with albumin.
• In electrophoresis, resembles pattern of
serum ie all proteins are present.

35.  Glomerular proteinuria causes
• Glomerulonephritis
• Minimal change disease
• Preeclampsia
• Strenous exercise, fever
• orthostatic or prostural proteinuria
 Postural (orthostatic) proteinuria
• due to lordotic posture that causes inferior venacaval
compression between the liver and vertebral column.
• Amount of proteinuria is <1000 mg/day.
• First-morning urine after rising is negative for proteins, while
another urine sample collected after patient performs normal
activities is positive for proteins.

36.  Tubular proteinuria due to excretion of low
molecular weight proteins like β2-microglobulin,
retinol-binding protein, lysozyme, α1-microglobulin,
and free immunoglobulin light chains
e.g. acute and chronic pyelonephritis, heavy metal
poisoning, tuberculosis of kidney, interstitial nephritis,
cystinosis, Fanconi syndrome and rejection of kidney
transplant.
Urine electrophoresis show prominent prominent α- and
β-bands (where low molecular weight proteins migrate)
and a faint albumin band.

37.  Post-renal proteinuria: This is caused by inflammatory
or neoplastic conditions in renal pelvis, ureter, bladder,
prostate, or urethra.

38. CHEMICALEXAMINATION
Test for Proteins
Heat and acetic acid test
 Principle –
 Proteins get precipitated when boiled
in acidic solution.
 Method –
 Take a 5 ml 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).
 Compare the heated part with the
lower part.
 Turbidity indicates presence of
either proteins or phosphates
 If precipitation or turbidity appears, add a few drops of 10% acetic acid.

39. CHEMICALEXAMINATION
Test for Proteins
• 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.

40. CHEMICALEXAMINATION
Test for Proteins
Reagent Strip Method
 Principle:
 Indicator strip coated with bromo-phenol blue indicator
 Buffered to pH 3.0 withcitrate
 Protein changes ionization ofdye which lead to change in
color of strip.
 Intensity of color is directly proportional to
concentration of protein.
 Semi quantitative method
 Screening method.
 Notes:
 Mainly reactive toalbumin
 False negative- Bence Jones protein, myoglobin,
haemoglobin
 False positive- Gross haematuria, highly alkaline
urine, contaminated urine

41. CHEMICALEXAMINATION
Test for Proteins
 Sulphosalicylic Acid Test
 Principle –
 Addition of sulphosalicylic acid to the urine
causes formation of a white precipitate if
proteins are present.
 Method –
 Take 2 ml of clear urine in a test tube.
 If reaction of urine is neutral or alkaline, a
drop of glacial acetic acid is added.
 Add 2-3 drops of sulphosalicylic acid (3 to
5%)
 See for turbidity against a dark background

42. CHEMICALEXAMINATION
Test for Proteins
Sulphosalicylic Acid Test
Notes:
 More sensitive and reliable than boiling test
 False positive- gross haematuria
highly concentrate urine
radiographic dye
excess uric acid
tolbutamide,sulphonamide, salicylates, penicillin
 False negative- very diluteurine
 Can detect- albumin, myoglobin, haemoglobin, BenceJones protein.

43. CHEMICALEXAMINATION
Test for Proteins
Quantitative Estimation of Proteins
 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
Proteinuria >1500 mg/ 24 hours indicates glomerular disease
Proteinuria >3500 mg/24 hours is called as nephrotic range proteinuria
Proteinuria is usually < 1500 mg/ 24 hours in tubular, hemodynamic
and post renal diseases

44. CHEMICALEXAMINATION
Test for Proteins
Methods for quantitation of proteins.
1. Quantitative estimation of proteins in a 24-hour urine
sample:
Various methods are available for quantitative estimation of
proteins:
Esbach’s albuminometer method,
turbidimetric methods,
biuret reaction and immunologic methods.

45. 2. Estimation of protein/creatinine ratio in a random urine
sample:
 This is done because of incomplete 24 hr urine collection.
 Normal protein/creatinine ratio is < 0.2.
 In low-grade proteinuria - 0.2-1.0;
 In moderate, it is 1.0-3.5; and
 In Nephrotic- range proteinuria it is > 3.5.

46. CHEMICALEXAMINATION
Microalbuminuria
 Urinary excretion of 30 to 300 mg/24 hours 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.

47. CHEMICALEXAMINATION
Bence 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).

48. CHEMICALEXAMINATION
Test for Bence Jones Proteinuria
•This test is not specific for Bence Jones proteins and both false-positive
and negative results can occur.
•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
 Upon heating, 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, there is reappearance of precipitate of Bence Jones
proteins.

49. CHEMICALEXAMINATION
Glucose
 Glucose is reabsorbed by the proximal renal tubules and
returned to circulation.
Normal threshold for glucose: 180-200mg%
When blood sugar level crosses this level
Detectable amounts of sugar in urine

50. CHEMICALEXAMINATION
Glucose
 Causes of Glycosuria:
1. Glycosuria with hyperglycemia:
 Endocrine diseases: Diabetes mellitus
Acromegaly
Cushing’s syndrome
Hyperthyroidism
Pancreatic disease.
 Non-endocrine diseases: central nervous system
diseases
 Drugs: adrenocorticotrophic hormone, corticosteroids,
thiazides.

51. CHEMICALEXAMINATION
Glucose
2. Glycosuria without hyperglycemia: Renal glycosuria.
 Renal threshold is the highest glucose level in blood at which
glucose appears in urine and which is detectable by routine
laboratory tests.
 Normal renal threshold for glucose is 180 mg/dl.
 In this disease, renal threshold is set below 180 mgs/dl but
glucose tolerance is normal
 The disorder is transmitted as autosomal dominant.

52. CHEMICALEXAMINATION
Test for Glucose
 Benedict’s qualitative test:
 Composition of Benedict’s qualitative reagent:
Copper sulphate (17.3gm)
Sodium carbonate (100gm)
Sodium citrate (173gm)
Distilled water (1000ml)
 Principle – heat
 Cupric ions + reducing sugar Cuprous oxide + cuprous hydroxide
Detects all reducing substances like glucose, fructose ,lactose
 Screening test.

53. CHEMICALEXAMINATION
Test for Glucose
Benedict’s qualitative test
 Method:
5ml of Benedict’s reagent +8 drops of urine
Boil themixture.
COLOR CHANGE
 Nil: no change from the Blue color
Trace: Green without precipitate
 1+(approx 0.5grams/dl)Green with precipitate
 2+(approx 1.0 grams /dl) Brownprecipitate
 3+(approx 1.5 grams /dl) Yellow orangeprecipitate
 4+(approx >2grams /dl ) Red precipitate

54. CHEMICALEXAMINATION
Test for Glucose
 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.
 Two methods used are two drop method and five drop
method.

55. CHEMICALEXAMINATION
Test for Glucose
Reagent strip method
 This test is specific for glucose.
 It is based on glucose oxidase-peroxidase reaction.
 Reagent area - two enzymes - glucose oxidase and
peroxidase and a chromogen.

56. CHEMICALEXAMINATION
Test for Glucose
Reagent strip method.
 The strip is dipped into the urine sample and color is
observed after a specified time and compared with the
color chart provided.
 Specific only for glucose.
 False positive test -oxidizing agent which oxidizes the
chromogen directly.
 False-negative test -ketones, salicylates, ascorbic acid,
and severe Escherichia coli infection.

57. CHEMICALEXAMINATION
Test for Glucose
Additional Tests for Sugars
 Fructose -thin-layer chromatography. A qualitative measurement,
a resorcinol test, is useful.
 Galactose Thin-layer chromatography. However, the disease is
usually identified by erythrocyte enzyme assay when suspected.
 Lactose -thin-layer chromatography or a qualitative lactose test
• Procedure-
To 15 mL urine in a test tube + 3 g lead acetate.
Shake and filter.
Boil filtrate + add 2 mL concentrated NH4OH and boil
brick-red solution due to lactose
red precipitate with clear supernatant

58. CHEMICALEXAMINATION
Test for Glucose
Additional Tests for Sugars
 Pentose: thin-layer chromatography.
 Sucrose: Sucrose will ferment yeast and can be separated by
chromatography.

59. CHEMICALEXAMINATION
Ketones
Defective carbohydrate metabolism /Deficient carbs in diet
Metabolism of fatty acids increases
Incomplete fat metabolism
Ketones in blood and urine
ACETONE (2%)
ACETOACIDIC ACID (20%)
beta HYDROXYBUTYRIC ACID (78%)

60. CHEMICALEXAMINATION
Causes of Ketonuria
Decreased utilization of carbohydrates:
1. Uncontrolled diabetes mellitus with ketoacidosis
2. Glycogen storage disease (von Gierke’s disease)
Decreased availability of carbohydrates in the diet:
1. Starvation
2. Persistent vomiting in children
3. Weight reduction program (severe carbohydrate restriction with normal fat intake)

61. CHEMICALEXAMINATION
Causes of Ketonuria
 Increased metabolic needs:
a. Fever in children
b. Severe thyrotoxicosis
c. Pregnancy
d. Protein calorie malnutrition

62. CHEMICALEXAMINATION
Tests For Ketone Bodies
Rothera’s test
 Principle:
Acetoacetic acid / acetone + sodium nitroprusside
purple color
 Method:
5ml of urine + ammonium sulphate + sodium nitroprusside.
slowly run ammonia along the sides of the test tube.
purple colored ring
 False-positive test can occur in the presence of L-dopa in urine
and in phenylketonuria.
Alk soltn

63. CHEMICALEXAMINATION
Tests For Ketone Bodies
Acetest tablet test
 The Acetest tablet consists of sodium nitroprusside,
glycine, andan alkaline buffer.
A purple lavender discoloration of the tablet
indicates thepresence of acetoacetate or acetone (≥ 5
mg/dl).
Gives a rough estimate of ketone bodies

64. CHEMICALEXAMINATION
Tests For Ketone Bodies
Ferric chloride test (Gerhardt’s)
10 % ferric chloride + acetoacetic acid in urine reddish /
purple color
 False positive - salicylate and dopa
 Sensitivity of the test - 25-50 mg/dl.

65. CHEMICALEXAMINATION
Tests For Ketone Bodies
Reagent strip test
 Modifications of nitroprusside test.
 Sensitivity -5-10 mg/dl of acetoacetate.
 False-negative – if exposed to moisture
 Ketone pad on the strip test is especially vulnerable to
improper storage and easily gets damaged.

66. CHEMICALEXAMINATION
Bile Pigment (Bilirubin)
Unconjugated bilirubin is not water-
soluble, is bound to albumin, and cannot
pass through the glomeruli. Therefore, it
does not appear in urine
Conjugated bilirubin is water soluble, is
filtered by the glomeruli, and therefore
appears in urine.
 Bilirubin (a breakdown product of hemoglobin) is
undetectable in the urine of normal persons.
 Presence of bilirubin in urine is called as
bilirubinuria.
After its formation from
hemoglobin in
reticuloendothelial
system

67. CHEMICALEXAMINATION
Bile Pigment (Bilirubin)
Detection of bilirubin in urine (along with urobilinogen)
is helpful in the differential diagnosis of jaundice
Urine test Hemolytic
Jaundice
Hepatocellular
Jaundice
Obstructive
Jaundice
Bilirubin Absent Present Present
Urobilinogen Increased Increased Absent
•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).

68. CHEMICALEXAMINATION
Tests For Detection of Bilirubin in Urine
1. Foam test:
 About 5 ml of urine in a test tube is shaken and observed for
development of yellowish foam.
 In normal urine, foam is white.
2. Gmelin’s test:
3 ml of concentrated nitric acid + equal quantity of urine
Shake the tube gently
Play of colors (yellow, red, violet, blue, &
green) indicates +ve test.

69. CHEMICAL EXAMINATION
Tests for Detection of Bilirubin in Urine
3. Lugol iodine test:
Lugol iodine solution-
• iodine 1 gm
• potassium iodide 2 gm
• distilled water to make 100 ml
4 ml of lugols iodine solution + 4 drops of urine.
(MIX)
GREEN COLOR – POSITIVE TEST

70. 4. Fouchet’s test:
A simple and sensitive test.
2.5 ml of 10% of barium chloride + 5 ml of fresh urine in a
test tube and mix well. A precipitate of sulphates appears to
which bilirubin is bound (barium sulphate-bilirubin
complex).
i. Filter to obtain the precipitate on a filter paper.
ii. add 1 drop of Fouchet’s reagent (25 g of trichloroacetic
acid, 10 ml of 10% ferric chloride, and distilled water 100
ml).
iii.Immediate development of blue-green color around
the drop indicates presence of bilirubin.
CHEMICALEXAMINATION
Tests For Detection of Bilirubin in Urine

71. CHEMICALEXAMINATION
Tests For Detection of Bilirubin in Urine
5. Reagent strips or tablets impregnated with diazo
reagent:
 Based on reaction of bilirubin with diazo reagent.
 The color change is proportional to the concentration of
bilirubin.
 Tablets (Ictotest) detect 0.05-0.1 mg of bilirubin/dl of
urine.
 The reagent strip tests are less sensitive (0.5 mg/dl).

72. CHEMICALEXAMINATION
Bile Salts
 Bile salts are salts of bile acids: cholic,
deoxycholic, chenodeoxycholic, and
lithocholic.
 Bile salts along with bilirubin can be detected in
urine in cases of obstructive jaundice.
 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.

73. CHEMICALEXAMINATION
Tests For Bile Salts
Hay’s surface tension test :
 Take some fresh urine in a conical glass tube.
 Sprinkle particles of sulphur.
 Sulphur particles sink to the bottom - bile
salts are present
 Sulphur particles remain on the surface of
urine- bile salts are absent.
 Thymol (used as a preservative) gives
false positive test.

74. CHEMICALEXAMINATION
Urobilinogen
 Urobilinogen is colorless; upon oxidation it is
converted to urobilin, which is orange-yellow in
color.
 Normally about 0.5-4 mg of urobilinogen is
excreted in urine in 24 hours.
 Urinary excretion of urobilinogen shows
diurnal variation with highest levels in
afternoon.
 A 2-hour post-meal sample is usually preferred.

75. CHEMICALEXAMINATION
Urobilinogen
Causes of Increased Urobilinogen in Urine:
1. Hemolysis:
 Increased urobilinogen in urine without bilirubin
 Excessive destruction of red cells leads to hyperbilirubinemia and
therefore increased formation of urobilinogen in the gut.
2. Hemorrhage in tissues:
 There is increased formation of bilirubin from destruction of red cells
Causes of Reduced Urobilinogen in Urine
1. Obstructive jaundice:
In biliary tract obstruction,delivery of bilirubin to the intestine is restricted
And very little or no urobilinogen is formed. This causes clay colored stools
2. Reduction of intestinal bacterial flora-
This prevents conversion of bilirubin to urobilinogen in the intestine.
It is observed in neonates and following antibiotic treatment

76. CHEMICALEXAMINATION
Tests For Urobilinogen
Ehrlich’s aldehyde test
 Ehrlich’s reagent (p-dimethylaminobenzaldehyde) reacts with
urobilinogen in urine to produce a pink color.
 Ehrlich’s aldehyde reagent -hydrochloric acid 20 ml
distilled water 80ml
paradimethylaminobenzaldehyde 2 gm
5 ml of fresh urine +0.5 ml of Ehrlich’s aldehyde reagent
Allowed to stand at room temperature for 5 minutes.
Dark red color – increased amount of
urobilinogen

77. CHEMICALEXAMINATION
Tests For Urobilinogen
Reagent strip method:
 This method is specific for urobilinogen.
 Test area is impregnated with either p-
dimethylaminobenzaldehyde or 4-methoxybenzene
diazonium tetrafluoroborate.

78. CHEMICALEXAMINATION
Blood
 The presence of abnormal number of intact red blood cells in
urine is called as hematuria.
Causes of Hematuria
1. Diseases of urinary tract
• Glomerular diseases: Glomerulonephritis,
Berger’sdisease, lupus nephritis, Henoch-S.chonlein
purpura.
 Non-glomerular diseases: Calculus, tumor, infection,
tuberculosis, pyelonephritis, hydronephrosis, polycystic
kidney disease, trauma, after strenuous physical
exercise, diseases of prostate (benign hyperplasia of
prostate, carcinoma of prostate.
2. Hematological conditions:
 Coagulation disorders, sickle cell disease

79. CHEMICALEXAMINATION
Tests for Detection of Blood in Urine
1. Microscopic examination of urinary sediment:
 Microscopic hematuria is presence of 3 or more number of
red blood cells per high power field on microscopic
examination of urinary sediment in two out of three properly
collected samples.
2. Chemical tests: detect both intracellular and extracellular
hemoglobin as well as myoglobin.
Heme proteins in hemoglobin act as peroxidase, which reduces
hydrogen peroxide to water.
This process needs a hydrogen donor (benzidine,
orthotoluidine, or guaiac). Oxidation of hydrogen donor leads
to development of a color .
Intensity of color produced is proportional to the amount of
hemoglobin present

80. CHEMICALEXAMINATION
Tests for Detection of Blood in Urine
Chemical tests
 Benzidine test:
 Make saturated solution of benzidine in glacial acetic acid.
 Mix 1 ml of this solution with 1 ml of hydrogen peroxide in a test tube.
 Add 2 ml of urine.
 If green or blue color develops within 5 minutes, the test is positive.
 Orthotoluidine test:
 In this test, instead of benzidine, orthotoluidine is used.
 It is more sensitive than benzidine test.
 Reagent strip test:
 Various reagent strips are commercially available which use different
chromogens (o- toluidine, tetramethylbenzidine).

81. CHEMICALEXAMINATION
Tests for Detection of Blood in Urine
Chemical tests
Causes of false-positive tests:
• Contamination of urine by menstrual blood in females
• Contamination of urine by oxidizing agent (e.g. hypochlorite or
bleach used to clean urine containers), or microbial peroxidase in
urinary tract infection.
Causes of false-negative tests:
• Presence of a reducing agent like ascorbic acid in high concentration:
Microscopic examination for red cells is positive but chemical test is
negative.
• Use of formalin as a preservative for urine

82. CHEMICALEXAMINATION
HEMOGLOBIN
Presence of free hemoglobin in urine is called as hemoglobinuria.
Causes of Hemoglobinuria
1. Hematuria with subsequent lysis of red blood cells in urine of low specific
gravity.
2. Intravascular hemolysis: Hemoglobin will appear in urine when haptoglobin
(to which hemoglobin binds in plasma) is completely saturated with
hemoglobin. Intravascular hemolysis occurs in infections (severe falciparum
malaria, clostridial infection, E. coli septicemia), trauma to red cells (march
hemoglobinuria, extensive burns, prosthetic heart valves), etc.
Tests for Detection of Hemoglobinuria
Tests for detection of hemoglobinuria are benzidine test, orthotoluidine test, and
reagent strip test.

83. CHEMICALEXAMINATION
HEMOSIDERIN
 Hemosiderin in urine (hemosiderinuria) indicates
presence of free hemoglobin in plasma.
 Hemosiderin appears as blue granules when
urine sediment is stained with Prussian blue stain
 Granules are located inside tubular epithelial cells
or may be free if cells have disintegrated.
 Hemosiderinuria is seen in intravascular
hemolysis.

84. CHEMICALEXAMINATION
MYOGLOBIN
 Myoglobin is a protein present in striated muscle (skeletal and
cardiac) which binds oxygen.
 Causes of myoglobinuria include injury to skeletal or cardiac
muscle, e.g. crush injury, myocardial infarction, dermatomyositis,
severe electric shock, and thermal burns.
 Ammonium sulfate solubility test is used as a screening test for
myoglobinuria (Myoglobin is soluble in 80% saturated solution of
ammonium sulfate, while hemoglobin is insoluble and is
precipitated. A positive chemical test for blood done on
supernatant indicates myoglobinuria).

85. CHEMICALEXAMINATION
Distinction between hematuria,
hemoglobinuria, and myoglobinuria.
Parameter Hematuria Hemoglobinuria Myoglobinuria
Urine color Normal, smoky, red,
or brown
Pink, red, or brown red or brown
Plasma color Normal Pink Normal
Urine test based on
peroxidase activity
Positive Positive Positive
Urine microscopy Many red cells Occasional red cell Occasional red cell
Serum haptoglobin Normal Low Normal
Serum creatine
kinase
Normal Normal Markedly increased

86. CHEMICALEXAMINATION
Chemical 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. E.coli,Salmonella, Proteus, Klebsiella,
etc.) are present in urine, they will reduce the nitrates to nitrites through
the action of bacterial enzyme nitrate reductase.
 Nitrites are then detected in urine by reagent strip tests.
 Screening test for UTI.

87. 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.

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