The document discusses the physical and chemical examination of urine, including the indications, collection, storage, and preservation of urine samples as well as the various tests involved in urinalysis such as assessment of volume, appearance, specific gravity, and microscopic examination of sediment. The goal of urinalysis is to screen for and aid in the diagnosis of conditions affecting the urinary tract, kidneys, liver, and metabolic disorders by examining the physical, chemical, and microscopic properties of urine.

1. Physical and
Chemical
examinationof
Urine
Dr. Himil Parikh
First year resident
GCS Medical college, Ahmedabad
Guided by-
Dr. Deepak Joshi
Dr. Kalpen Patel

2. Introduction
• Urine can be used to look for physical and biochemical
abnormalities.
• To screen for and/or to aid in diagnosis of conditions
such as a urinary tract infections, kidney disorders,
liver problems, diabetes or other metabolic conditions,
to name a few.
• For example:
• Persistent amounts of albumin and other proteins in the urine
(proteinuria) indicate kidney damage.

3. Indications
1. Suspected RENAL DISEASES like
glomerulonephritis, nephrotic syndrome,
pyelonephritis, acute and chronic renal failure.
2. Detection of URINARY TRACT INFECTION.
3. Detection and monitoring of METABOLIC
DISORDERS like Diabetes mellitus.
4. Differential diagnosis of JAUNDICE.
5. Detection and monitoring of PLASMA CELL
DYSCRASIAS.
6. Diagnosis of PREGNANCY.

4. Collection of Urine
• Improper collection---- may invalidate the results
• Containers for collection of urine should be wide
mouthed, clean and dry.
Time of Collection
Single
specimen
24 hour
specimen
First voided specimen Random Postprandial specimen

5. First voided specimen:
Most concentrated and has acidic pH, so formed
elements (cells and casts) are well preserved. It is
preferred for urinalysis.
Random specimen:
Single specimen collected at any time of day and is
sufficient for routine urine examination (though not
preferred, it is the most frequently received
specimen)
Post-prandial specimen: (collected 2 hours after a meal in the
afternoon)
Sometimes requested for estimation of glucose or of
urobilinogen.

6. About 15 ml of midstream sample (especially in female
patients) is cleanly collected.
Collect clean catch sample.
24- hour specimen:
Quantitative estimation of proteins, hormones and
electrolytes

7. Collection Methods
• Midstream specimen
• Clean-catch specimen
• Timed collection
• Catheter specimen
Used for routine and bacteriological study in bedridden
patients.
• Infants
Either by attaching a clean plastic bag around baby’s genitalia
and leaving it there for some time or suprapubic puncture

8. Collection and Transport Guidelines
All urine collection and/or transport containers should
be:
1. Clean and leak proof.
2. Break-resistant
3. Material of container should not interfere and
4. Container should not be re-used.
5. Capacity of at least 50 ml (routine) and at least 3
litre (24-hour sample)
6. Amber coloured containers for light sensitive
analytes

9. Storage of urine
• Ideally the specimen should be examined within 2 hours
of voiding.
• If delay is expected, it should be refrigerated at 4-6°
Celsius for up to 8 hours.

10. Effect of Storage on urine:
1. Increase in pH: Due to production of ammonia from urea
by urease producing bacteria.
2. Formation of crystals: Precipitation of phosphate and
calcium.
3. Loss of ketone bodies: since they are volatile.
4. Decrease in glucose: Due to glycolysis and utilization of
glucose by cells and bacteria.
5. Oxidation of bilirubin to biliverdin: false-negative test for
bilirubin
6. Oxidation of urobilinogen to urobilin: false-negative test
for urobilinogen.
7. Bacterial proliferation
8. Disintegration of cellular elements (Especially in alkaline
and hypotonic urine.

11. Preservation of Urine Sample
• Not recommended for routine analysis as they interfere with
reagent strip techniques and chemical test for protein.
Preservatives for 24-hour urine sample:
1. Hydrochloric acid: Used when detecting adrenaline, nor-
adrenaline, vanillylmandelic acid (VMA) and steroids.
2. Toluene: It forms a thin layer and hence physical barrier against
bacteria and air.
3. Boric acid: General preservative (sample can be kept for 24
hours without refrigeration)
4. Thymol: Inhibits bacteria and fungi.
5. Formalin: Excellent for preservation of formed elements.

12. Urinalysis
The basic (routine) urinalysis consists of four parts:
1. Specimen evaluation
2. Physical examination
3. Chemical examination
4. Microscopy of urine sediment/ Sediment examination

13. Specimen
Evaluation
Urinalysis

14. Preanalytical Assessment
• Before proceeding for examination, specimen must be evaluated
in terms of its acceptability.
1. Minimum Labeling Requirements: Patient’s full name, Date
and Time of collection
2. Intactness: There shouldn’t be any leakage, spillage and damage
to container.
3. Timing of collection: First voided morning urine is the best for
Routine analysis.
4. Preferences: If multiple investigations are to be done from a
single specimen, bacteriologic examination should be
performed first. Hence, volume of urine should be noted
properly.

15. Physical
Examination
Urinalysis

16. Physical examination
It includes:
1. Volume
2. Appearance
3. Odor
4. Specific gravity

17. Volume
• Total volume can be evaluated only from 24-hour urine
sample.
• Main determinant of urine volume is water intake.
• Normal individual:
– 24-Hour urinary output: 600 to 2000 ml out of which
about 400 ml is produced during night
(Reference: Todd and Stanford)
Exceptions:
Pregnancy- diurnal variation may be reversed
Young children- 3-4 times more urine than adults (ml/per
body weight)

18. Increase in Urine
volume
Decrease in Urine
volume
>2000 ml/ 24 hours- Polyuria
>500 ml during night- Nocturia
Specific gravity- <1.018
<500 ml/ 24 hours- Oliguria
<100 ml/ 24 hours or complete
cessation- Anuria
Cause
s
Causes
•Diabetes mellitus (Osmotic diuresis)
•Diabetes insipidus (Failure to secrete
ADH)
•Chronic renal failure (Loss of
concentrating ability of renal tubules)
•Diuretic therapy
•Polydypsia
•Caffeine/ alcohol intake
• Oliguria:
High grade febrile states
Acute glomerulonephritis
(decreased glomerular filtration)
Congestive cardiac failure or
dehydration (Renal
hypoperfusion)
• Anuria:
Acute tubular necrosis
Complete urinary obstruction

19. Appearance
Colour Clarity
Normally, urine is AMBER coloured and CLEAR
Colour Pathological Non pathological
White Chyle
Pus
Phosphates
Admixture of semen (in males)
Yellow to Orange Bilirubin
Urobilin
Concentrated urine
Vit- B Complex therapy
Rifampicin
Acriflavine
Pyridium
Carrots
Senna
Pink to Red Haemoglobin
Myoglobin
Porphyrins
Red blood cells
Beets(anthocynin)
Aminopyrine
Methyldopa
Food color

20. Colour Pathological Nonpathological
Red to Brown to Purple Porphobilinogen
Uroporphyrin
Brown to Black Homogenistic acid
Melanin
Methaemoglobin
(Black-water fever)
Phenol
Porphyrins
Iron compounds
Chloroquine
Levodopa
Metronidazole
Quinine
Blue to Green Biliverdin
Pseudomonas
infection
Methylene blue
Vit B complex
Phenyl salicylate
Amitryptiline

21. Clarity
Non- Pathologic
• Turbidity due to precipitation of crystals or non-
pathologic salts known as AMORPHOUS
In alkaline
urine
In acidic urine
Precipitation of-
•Phosphate
•Ammonium urate
•Carbonate
Redissolves on acidification
Precipitation of-
•Uric acid
•Urates
Redissolves on warming at 60°

22. Pathologic
Leukocytes Bacteria Chyluria Lipiduria
Cloudy urine
(just like
phosphate
containing urine)
which doesn’t
dissolve on
acidification
Uniform
opalescence
which will
remain even
after
acidification or
centrifugation.
Milky Urine -
contains lymph
due to rupture of
lymph vessels
into urinary tract
E.g. Filariasis
Pseudochyluria
occurs with use of
paraffin based
vaginal creams in
treatment of
Candida infection
Milky Urine -
Seen with
Nephrotic
syndrome
(contains TGLs
and cholesterol)
Also in
sustained
skeletal
trauma with
fractures to
major long
bone
* Smoky urine - RBCs
* Hazy urine - Mucous

23. Ether extraction
Extracted Not Extracted
Chyluria Pseudochyluri
a
Lipiduri
a
Triglycerides
Yes No
Differentiating Chyluria from
Lipiduria

24. Odour
• Freshly voided urine- Typical aromatic odour (Volatile organic
acids)
• On standing- Conversion of urea  ammonia by urease
producing bacteria – Faint ammoniacal odour
Odour Cause
Fruity Ketonuria
Mousy Phenylketonuria
Fishy UTI by Proteus, Tyrosinemia
Ammoniacal UTI with E. coli, Old standing urine
Foul Urinary Tract Infection (UTI)

25. Odour Cause
Maple syrup Maple syrup urine disease (MSUD)
Rancid Tyrosinemia
Cabbage Methionine malabsorption
Sweaty feet Isovaleric acidemia and glutaric acidemia
Rotten eggs Cystinuria

26. Specific Gravity
• Also known as Relative mass density
• Ratio of weight of a volume of urine to the weight of
the same volume of distilled water at a constant
temperature.
• Main contributors to specific gravity of urine are urea and
sodium
• Measures the concentrating and diluting power of kidney.
Concentrating ability of kidney is one of the first
function to be lost as a result of tubular damage.
• Normal range of random specimen-1.003-1.035
• Range of 24 hour sample in Normal adults with
adequate fluid intake- 1.015- 1.025.

27. Specific gravity and
Osmolality are not the same!
Specific Gravity:
•Indicates relative proportions
of dissolved solid components
to total volume of the specimen
i.e. DENSITY of urine
•Larger particles like glucose
and protein tend to increase
the Sp.Gr. more than smaller
electrolytes
Osmolality:
•Indicates number of particles
of solute per unit of solution
•No difference occurs in the
results with different sized
particles i.e. glucose and protein
OR smaller electrolytes
* In certain circumstances, measurement of Osmolality is
preferred over measurement of Specific gravity.

28. Increased specific gravity
• Diabetes mellitus
• Dehydration
• Proteinuria (Nephrotic
syndrome)
• Glycosuria
Decreased specific gravity
 Polydipsia
 Diabetes insipidus
 Diuretics
 Early stages of Chronic
Kidney Disease
Causes:
*Hyposthenuria: Consistently low specific gravity; <1.007
*Hypersthenuria: Consistently high specific gravity
*Isosthenuria: Fixed specific gravity at 1.010 (occurs in Chronic Kidney
Disease)

29. Methods of Specific Gravity
measurement
1. Reagent strip method- Most commonly employed
2. Urinometer
3. Refractometer
4. Falling drop method- Least commonly performed
INDIRECT METHODS
DIRECT METHODS

30. Reagent Strip method
• Has 3 main ingredients:
• PRINCIPLE: pKa change of pretreated polyelectrolyte
in relation to the ionic concentration of urine.
• Higher the concentration of urine, lower the pKa as pKa
refers to the proton donating capacity of an acid and
stronger the acid, lower the pKa.
1. Polyelectrolyte
2. Indicator substance
3. Buffer

31. Advantages:
1. This method is not affected by the high amounts of protein and
sugar and so there is no need for the correction of value.
2. This is a method unaffected by Temperature changes.
Disadvantages:
• False readings may be obtained if there is a run-over from
adjacent reagent area in excessively wetted strips.

32. Urinometer
• Direct method of assessing specific gravity of urine at room
temperature which is based on the principle of BUOYANCY.
ADVANTAGES:
1. Results are directly obtained and depend on the solutes dissolved in
urine.
2. Easily performed and quick assessment of results
DISADVANTAGES:
1. Requires minimum of 15 ml for measuring specific gravity
2. Correction of 0.001 should be made for each 3° C rise or fall in
temperature.
3. Correction for every g/dl of protein and sugar is required (0.003
for 1 g/dl of protein and 0.004 for 1 g/dl of glucose)

33. URINOMETER

34. Refractometer
• Indirect method related to content of dissolved solids present
• It is obtained by ratio of velocity of light in air to the velocity of
light in a solution
• ADVANTAGES:
• DISADVANTAGES:
1. Only a few drops of urine are required
2. The results obtained are quite accurate
1. Valid only for Urine
2. Damaged by heat above 150° F and by
immersion of eyepiece and focusing
ring in water
3. Requires daily calibration for accurate
results

35. Falling drop method
• More accurate and precise than both Urinometer and
Refractometer
• A drop of urine is allowed to fall in a specially designed
column.
• This drop encounters two beams of light, first one starts
the timer and the second one stops it. This falling time is
measured electronically and expressed as specific gravity.

36. Chemical
Examination
Urinalysis

37. Chemical examination of urine
• Reagent strips are the primary method used for the chemical
examination of urine.
• They represent multiple complex, state-of-the-art chemical reactions.
Advantages of Multistix Strip
• Quick screening of urine chemistry
• Reliable, specific and sensitive
• Avoids use of various corrosive reagents, different type of glass wares
and other laboratory material required for wet chemical testing of urine
• It can be performed in uncentrifuged urine and doesn’t require
acidification
• Less labour intensive and can be automated for large laboratories.
• Less chance of human error

38. Reaction/ pH
• pH is the scale for measuring acidity or alkalinity
• On standing, urine becomes alkaline due to formation of
ammonia from urea and loss of CO2. Hence, freshly
voided urine specimen is required.
• Methods:
• Normal pH is 4.6 to 8.0
1. Reagent strip method
2. Litmus paper test
3. pH meter

39. Reagent strip method
• Indicators methyl red and bromothymol blue give a range of
orange, green, and blue colours as the pH rises, permitting
estimation of pH values to within half a unit within the range of 5
to 9.
Acidic urine Alkaline urine
Diabetes mellitus UTI by urea splitting
organisms (Proteus and
Pseudomonas)
Starvation Severe vomiting
Fever Vegetarian diet (Citrus fruits)
UTI by E. coli Old ammoniacal urine sample
High protein diet Chronic renal failure

40. Problems:
• If the strips get excessively wet, acid buffer from the protein patch
runs into the pH patch, causing it to become orange.
• Results are not precise unless the test is performed on freshly voided
urine as with time, the pH increases with formation of ammonia and
release of CO2.
Reagent Strip

41. Other Methods:
• Litmus paper test: Blue litmus paper turns red, in
acidic urine and Red litmus turns blue in alkaline urine.
Not very precise, not preferably used
• pH meter: Electrode of pH meter is dipped in urine and
the reading is taken. It is used when exact pH is required.

42. • Uses:
1. To evaluate Renal acidosis/alkalosis and
compensatory mechanism activation in
pulmonary acidosis/alkalosis.
2. Evaluation of Tubular function (Exchange of
Anions and Cations)
3. Helps identify crystals in urine
4. When alteration of urinary pH is required in
treatment of stones
5. Certain drugs work better at a certain pH (For
e.g.: Streptomycin is effective in UTI if urine is
kept alkaline)

43. Protein in Urine
• Normal- Up to 150 mg/24 hours in a concentration varying from
2 to 10 mg/dl
• Tamm-Horsfall glycoprotein(urumucoid) secreted by the distal
tubular cells and ascending loop of Henle. It constitutes 1/3rd or
more of the total urinary protein loss.
• Protein has a very low maximal tubular rate of reabsorption
meaning increased filtration of protein quickly saturates the
reabsorptive mechanism.
• Hence, detection of abnormal amounts of protein- IMP indicator
of Renal disease

44. Common screening tests
• Qualitative/Semiquantitative
1. Colorimetric Reagent strip test
2. Precipitation-based testing
• Reagent Strip-
a) This method takes advantage of the protein error of pH
indicators. Because proteins carry a charge at physiologic pH,
their presence will elicit a pH change.
b) The reagent strip is impregnated with tetrabromphenol blue
buffered to an acid pH of 3 OR tetrachlorophenol
tetrabromosulfophthalein.
c) Variable shades of green develop if protein is present in urine.

45. Advantage:
• Advantage of avoiding false-positive reactions with organic
iodides (e.g. Radiograph contrast and tolbutamides or other drugs).
• Decreased chance of Human error
• Rapid results
Drawbacks:
• Lack of sensitivity of the reagent strip to globulins
• False positive results can occur with highly pigmented urine,
quaternary ammonium compounds, amidoamines in fabric
softeners, chlorhexidine, and excessive leaching of the acid buffer
of the test strip by excessive wetting.
• Highly buffered alkaline urine will also provide false-positive
results.

46. Reporting:
• Most methods will detect 5 to 20 mg of albumin per decilitre
• Results can be read in a “plus” system as negative, trace, and 1+
to 4+

47. Protein quantification
• The quantification of urine can be done by
obtaining 24 hour urine sample
Normal Protein: Creatinine ratio values are:
<0.2 g Protein per g of Creatinine in Adults and Children >2 years
<0.5 g Protein per g of Creatinine in Children <2 years
Quick Quantification
•When 24 hour sample is not available/feasible,
Protein: creatinine ratio can be used to estimate renal
function.

48. MINIMAL
PROTEINURIA
(<1 gm/day)
 Exercise
 Fever
 Emotional stress
 Chronic Pyelonephritis
 Nephrosclerosis
 Chronic interstitial nephritis
 Postural proteinurias
 Transient proteinurias
MODERATE
PROTEINURIA
(1-4 gm/day)
 Nephrosclerosis
 Toxic nephropathies
 Degenerative, Malignant,
Inflammatory conditions of
Lower urinary tract
 Irritative conditions of Lower
urinary tract like Calculi
 Pre-eclampsia
 Multiple myeloma

49. MARKED PROTEINURIA
(> 4 gm/day)
Nephrotic syndrome
Acute glomerulonephritis
Chronic glomerulonephritis, severe
Diabetic nephropathy, severe
Renal amyloidosis
Lupus nephritis
Toxemia of pregnancy
End Stage Renal Disease

50. FUNCTIONAL PROTEINURIA
 Usually less than 0.5 g/day
 Seen with dehydration and strenuous exercise
 Also with Congestive heart failure, cold exposure and fever
TRANSIENT PROTEINURIA
Proteinuria only on certain occasions and otherwise normal
Requires 6 monthly follow-up.

51. Sulfosalicylic Acid Method (Qualitative):
• Depends on formation of a precipitate on addition of 3%
Sulfosalicylic acid for determination of the presence of protein.
• This method will detect about 5 to 10 mg/dl. Albumin, globulins,
glycoproteins, and Bence Jones proteins are all detected.
* There are Quantitave methods available like Sulfosalicylic acid
method and Trichloroacetic acid method but these
quantitative methods have been found unsatisfactory.
Confirmatory test

52. Glucose and Other Sugars in Urine
• The presence of detectable amounts of glucose in urine is termed
glycosuria.
• Glucose may appear in the urine at different blood glucose levels,
and there is not always a concomitant hyperglycemia.
• Glycosuria usually occurs when the blood level is greater than
180 - 200 mg/dl (Reference: Henry’s Clinical Diagnosis and Management by Laboratory Methods –
23rd edition)
Causes of Glycosuria:
1. Renal Glycosuria (due to low renal threshold –
Acquired/Congenital)
e.g. Fanconi’s Syndrome
2. Gestational Glycosuria (Reduced Renal threshold)
3. Alimentary glycosuria – On consuming high amounts of
carbohydrates

53. Methods of Glucose estimation
1. Reagent strip method
2. Copper reduction tests
•Normally, 50 mg of disaccharides are excreted in 24 hours.
•With severe Intestinal diseases like sprue or acute enteritis,
this level may rise to 250 mg or more.

54. Reagent Strip method
• Principle: Based on a specific glucose oxidase and peroxidase
method, a double sequential enzyme reaction; reagent strips differ
only in the chromogen used.
• Chemistry:
Glucose + O2 Gluconic acid + H2O2
H2O2 + Chromogen Oxidized chromogen +
H2O
Glucose Oxidase
Peroxidase
*Multistix- potassium iodide chromogen- Color changes from blue to
brown at 30 seconds.

55. Drawbacks:
False-positive:
If strongly oxidizing cleaning agents are present in the urine
container.
Low specific gravity may falsely elevate results.
False-negative:
Use of Sodium fluoride as a preservative
High specific gravity
Occasionally by Ascorbic acid

56. Cross-verification
Copper Reduction Tests:
• If false-negative is suspected due to ascorbic acid, should be
confirmed with Copper reduction test
• In case of neonates and infants where excretion of sugars
other than glucose is suspected, copper reduction test is
mandatory.
• In those instances in which the copper method is positive and the
glucose oxidase method is negative, glycosuria is ruled out.
• Out of all Copper Reduction Tests, Benedict’s test is the most
sensitive test.
• Other tests include: 5-drop and 2-drop Clinitest tablet methods

57. Ketones in Urine
• Defect in carbohydrate metabolism or absorption or an inadequate
amount of carbohydrate in the diet- the body compensates by
metabolizing increasing amounts of fatty acids.
• In ketonuria, the three ketone bodies present in the urine are
acetoacetic (diacetic) acid (20%), acetone (2%), and
3-hydroxybutyrate (about 78%).
Acetoacetic acid Acetone
Acetoacetic acid 3-Hydroxybutyrate
- CO2
+2H
-2H
(Irreversible)
(Reversible)

58. Causes of Ketonuria
1. Diabetic Ketoacidosis:
Seen more commonly and more importantly with Type 1 Diabetes
mellitus.
2. Nondiabetic ketonuria:
Acute febrile diseases in infants and children, Hypoalimentation (e.g.
starvation), Hyperemesis of pregnancy, in cachexia and following
anesthesia.
3. Lactic Acidosis:
Can coexist with Diabetes mellitus, Renal failure, Liver disease.

59. Methods:
1. Reagent Strip method
2. Nitroprusside Tablet test
3. Gerhardt ferric chloride test (Only for acetoacetic acid)
4. Test tube nitroprusside method of Rothera
Commonly used method is the Reagent strip method as it is more
sensitive in detecting as low as 10 mg of acetoacetic acid.
(Less sensitive to Acetone)
None of the tests mentioned above detects Beta-Hydroxybutyric acid

60. Reagent Strip method
Principle: Based on nitroprusside reaction for ketone.
• The multistix strip contain buffers and sodium nitroprusside
which reacts with acetoacetic acid producing a pink maroon
colour in 15 seconds. It is less sensitive to Acetone.
• Reagent strip without alkali react to acetoacetate not acetone.
• The chemo strip reagent strip contain sodium nitroprusside
buffer and glycine which reacts with acetoacetic acid in presence
of alkaline medium to form violet dye.
• Sensitivity: 10 mg/dl of acetoacetic acid and 70 mg/dl of
acetone.

61. • Positive result is indicated by change in colour from beige to
violet within 60 seconds. It is not sensitive to gamma
hydroxybutyric acid.
• With large results like 3+, urine may be diluted and reassessed.
• Drawbacks
a) False positive is seen after the use of phthaleins or in presence
of extremely large amount of phenyl ketones , l-dopa and
8-hydroxyquinolone metabolites
b) False negative results occur because of loss of reagent.
Reaction may also not occur if specimen handling is improper
which causes acetone loss due to its volatile characteristic or
consumption by bacteria.

62. Other test
Rothera’s Test
• Saturate about 5ml of urine with ammonium sulphate.
• Add few crystals of sodium nitroprusside and shake well.
• Add liquor ammonia through the sides of the test tube.
• Formation of a purple ring at the junction indicate a positive test.
* Not a sensitive test and hence gives False-negative even in presence
of Ketones in urine.

63. Determination of Bilirubin
• Bilirubin is breakdown product of Hemoglobin which occurs in
Reticuloendothelial cells of Spleen, Liver and Bone marrow.
• The unconjugated Bilirubin thus formed is transported to liver for
conjugation with glucuronic acid to form Bilirubin glucuronide.
• Normal adult urine has only 0.02 mg/dl of Bilirubin
• Increased urinary Bilirubin occurs in-
1. Obstruction to bile outflow from liver
2. Hepatocellular disease with decreased excretion of conjugated
Bilirubin in bile
* Presence of Bilirubin in urine means presence of only CONJUGATED
BILIRUBIN

64. Methods:
1. Reagent Strip method
2. Diazo tablet method
3. Wash-through tablet method
• The reagent strip is much less reactive to free bilirubin than is
the tablet test so that a difference in results becomes more apparent
as the urine ages.
• The above point means that when urine is kept for a longer period
of time(especially when exposed to light), bilirubin glucuronide
gets converted free bilirubin which isn’t picked up accurately on
the Reagent strip but is seen with the Tablet test
• Hence, Tablet tests are confirmatory tests

65. Principle: The test is based on coupling reaction of bilirubin with
diazonium salt in strongly acidic medium.
• Multistix reagent strip: If bilirubin is present color changes from
cream buff to tan which is read at 20 seconds .This uses the
diazonium salt- diazotized 2,4-dichloroaniline.
• Detects 0.8 mg/dl of bilirubin
• Chemo strip reagent: Contains 6-dichlorobenzene diazonium
tetraflouroborate as salt which changes color from pink to violet
at 30 -60 seconds.
• Detects 0.5 mg/dl of bilirubin
Reagent Strip method

66. Drawbacks:
• Urine must be fresh because bilirubin glucuronide in urine
quickly hydrolyses to less reactive free bilirubin.
• False negative: Seen when specimen stands too long due to
oxidation of bilirubin. Large amount of ascorbic acid and
nitrite can also lower bilirubin results
• False positive: Rifampicin and large amount of chlorpromazine
metabolites may cause false positive
* Salicylates and urobilinogen do not interfere with the reaction.

67. Confirmatory Bilirubin Test:
Diazo tablet method:
• The reagent strip test is much less reactive to free bilirubin than
is the tablet test, so that a difference in results becomes more
apparent as the urine ages.
• The diazo test reacts positively to bilirubin in amounts as low as
0.05 to 0.1 mg per decilitre. No purple reaction is seen with
urobilin.

68. Bile salts and Bile pigments(BSBP)
Bile Salts (Hay’s Sulphur powder test):
• Bile salts have the property of decreasing surface tension.
• Hence, sulphur powder sprinkled to urine will settle down if bile
salts are present in urine.
Bile Pigments (Fouchet’s Test):
• Bile pigments adhere to the precipitates of barium sulphate.
• On addition of Fouchet’s reagent, ferric chloride in the presence of
trichloroacetic acid oxidizes yellow bilirubin to green biliverdin.

69. Determination of Urobilinogen
• Conjugated bilirubin reaches eventually to duodenum from liver
but without getting absorbed, passes on to colon where it is acted
upon by bacteria to form:
1. Urobilinogen
2. Mesobilirubinogen
3. Stercobilinogen
• Out of all these, only small amount of urobilinogen is excreted
in the urine after the enterohepatic circulation is complete.
• Normal output of urobilinogen in urine is 0.5 to 2.5 mg/24
hours

70. Causes of increased urobilinogen
excretion:
1. Hepatocellular damage due to viral hepatitis, drugs or toxic
substances or in some cases cirrhosis.
2. Congestive heart failure (Due to liver congestion, reexcretion
into bile is impaired).
3. Infection such as Cholangitis associated with obstruction
(Urobilinogen with bilirubin)
4. Hemolysis (Urobilinogen without bilirubin)- It can be one of the
indications for malaria infestation.

71. Methods:
1. Reagent Strip method
2. Ehlrich’s Aldehyde method
• For quantitative comparative purposes in the same patient, a 2-
hour test is used in which urine is collected from 2pm to 4pm after
lunch.
• This period coincides with heightened excretion of urobilinogen,
as the pH of the urine is more nearly neutral.
• Other 2-hour periods may be tested for comparison

72. Principle:
• The test is based on modified Ehrlich’s Aldehyde reaction .The
test area is impregnated with p-dimethyl aminobenzaldehyde in
conjunction with color ethanol which reacts with urobilinogen
in strongly acidic media to form yellow shades of red brown
(Multistix).
• This is not specific for urobilinogen .
• Other porphobilinogen, indole, sterol can also give same
color development.
• Chemo strip reagent: Test area is impregnated with 4-
methoxybenezene tetra flourate .This is specific for
urobilinogen.
Reagent Strip method

73. Drawbacks:
False negative:
• The test can be negative as urobilinogen is quite labile and will convert
to urobilin which is non-reactive therefore only fresh sample is
acceptable.
• If formalin is present it will give false negative result
False positives:
• Test can also be altered by p-aminosalicyclic acid, sulphonamide and p-
aminobenzoic acid.
• Bilirubin if present will give green color.

74. Indirect Tests for Urinary Tract
Infection
• Two most commonly utilized testing modalities for indirect
assessment of bacteriuria and leukocyturia are:
1. Reagent strip nitrite
2. Reagent strip Leukocyte esterase
• Microscopic urinalysis: Rapid confirmatory test for the
presence of leukocytes and bacteria
• Gold standard: Bacteriologic culture

75. Nitrite
• Detects 70% positive results when compared to cultures
• E-coli only: 93% agreement with cultures
Most common organisms causing positive nitrite test:
1. Escherichia coli
2. Klebsiella
3. Enterobacter
4. Proteus
5. Staphylococcus
6. Pseudomonas

76. Reagent Strip method
Principle:
• This is based on conversion of nitrate in urine to nitrite by the
action of bacteria like Proteus species, E.coli, Enterobacter, and
Klebsiella.
Nitrite+ p-arsinilic acid Diazonium complex
• This diazonium compound then couples with 1,2,3,4, tetra
hydrobenzoquinolin-3- ol to produce pink color.
• This indicates- 105. Or more organisms per ml.
* (Bacteriuria is >1,00,000 bacteria per ml of urine)
Acidic pH

77. • This method detects the presence of 0.075 gm of nitrite/dl.
Drawbacks:
• False positive is seen in stored sample due to contaminants and
post collection bacterial proliferation; and medication
(phenazopyridine)
• False negative result is seen due to ascorbic acid, urobilinogen or
low pH, lack of dietary nitrites.

78. Leukocyte Esterase
• Esterase activity- Marker for human neutrophils.
• Because neutrophils and other cells are labile in urine, leukocyte
esterase activity can be indicative of remnants of cells that are
not visible microscopically.
• Positive leukocyte esterase- Significant number of Neutrophils
either intact or lysed.
• It has a high negative predictive value.

79. Reagent Strip method
• Neutrophilic esterases catalyze the hydrolysis of esters to produce
their respective alcohols and acids.
Multistix:
• Utilizes 3-hydroxy-5- phenyl-pyyrole-N-tosyl-l-alanine ester as a
substrate, which reacts in the presence of leukocyte esterase to form
pyrrole alcohol.
• The alcohol then reacts with a diazonium salt to produce a purple
colour.
• The intensity of the colour produced is proportional to the amount of
enzyme present, which is related to the number of neutrophils present.

80. False negative:
• Seen in urine having-
1. High specific gravity,
2. High glucose concentration,
3. Drugs like gentamycin and high protein content
False positive:
• Contamination of urine with Vaginal fluids
• Trichomonas and eosinophils provide another source of
esterases and hence false positive results.

81. Blood, Hemoglobin, Hemosiderin,
and Myoglobin in Urine
• Hematuria: Presence of abnormal number of red blood cells in
urine.
• Hemoglobinuria: Presence of free hemoglobin in urine.
• Myoglobinuria: Presence of myoglobin in urine. For e.g.
musculoskeletal trauma

82. Hematuria
• Gross hematuria: Presence of increased number of red blood cells
so much so that it is visible to the naked eye
• Microscopic hematuria: 3 or >3 red blood cells per high-powered
field
• As it is important to identify red blood cells in urine and due to the
lysis of red blood cells in urine, some studies suggest that Reagent
strip method is more sensitive than urine microscopy to
identify hematuria.
• Yet, it is better to confirm this by microscopy of a fresh urine
specimen
* Alkaline pH and Low specific gravity can cause in-vitro lysis of Red Blood
Cells.

83. Causes of Hematuria
1. Diseases of Urinary tract:
Glomerular diseases: Glomerulonephritis, Berger’s disease, lupus
nephritis, Henoch-Schonlein purpura.
Non-glomerular disease: Calculus, tumour, infection, tuberculosis,
pyelonephritis, hydronephrosis, trauma, after strenuous exercise.
2. Hematological conditions: Coagulation disorders, Sickle
cell disease.
* Presence of Red cell casts and proteinuria along with hematuria suggests
glomerular cause of hematuria

84. Blood/ Heme proteins
Reagent Strip method
Principle:
• This method is based on the liberation of oxygen from
peroxide in reagent strip by peroxidase like activity of haem
in free haemoglobin of lysed RBC or myoglobin .
• Intact RBC are lysed on strip causing haemoglobin to react . The
reagent area is impregnated with buffered mixture of organic
peroxide and chromogen tetra methyl benzidine.
• Haemoglobin catalyses the oxidation of chromogen to produce
green colour which is read at 60 seconds.
• If green spot is present then intact RBCs are present
• If uniform green colour is present then it indicates the presence
of haemoglobin or myoglobin.

85. • Sensitivity is up to 0.05-3 Hb/dl of urine
False positive:
• Contamination of urine by menstrual blood in females.
• In presence of oxidizing agents (hypochlorite bacterial
content, microbial peroxidase associated with UTI)
False negative:
• Presence of ascorbic acid and formalin.
• Presence of nitrites in large amount will delay the
reaction.
* Confirmation of Hematuria can be done through Microscopy of Urine

86. GFR
• Glomerular filtration rate (GFR) is generally considered the
best overall indicator of the level of kidney function (Smith,
1951).
• Inulin clearance is widely regarded as the gold standard for
measuring glomerular filtration rate.
• Simpler or more practical method is creatinine clearance test.
Two approaches
Endogenous Exogenous
Creatinine
clearance
Inulin clearance

87. • Creatinine is the most widely used marker of GFR
for several reasons.
1. It is an endogenous substance with a fairly constant
rate of production.
2. Creatinine is not bound to plasma proteins, and
therefore is filtered freely by the glomerulus.
3. It is not reabsorbed by the renal tubules
4. Only a small amount is secreted by the tubules.

88. Estimated GFR (eGFR)
• In the Modification of Diet in Renal Disease (MDRD) study,
Levey and colleagues measured GFR by I125-iodothalamate to
derive a formula for estimating glomerular filtration rate using six
variables – age, sex, serum urea nitrogen, serum creatinine and
serum albumin concentration.
• This equation was simplified to include age, sex, race and serum
creatinine as follows:
GFR = 175 ×Cr × age ×1.212 (for black) × 0.742 (for
women)
−1.154 −0.203
Due to major limitation in accurate urine collection for measurement
of creatinine clearance, mathematical formulas have now been
developed to assess the GFR.

89. • Estimated GFR results are reported as ml/min/1.73m2
• A normal eGFR for adults is greater than 90 ml/min/1.73m2,
according to the National Kidney Foundation
• An eGFR below 60 ml/min/1.73m2 suggests that some kidney
damage has occurred
KIDNEY DAMAGE
STAGE
DESCRIPTION
ESTIMATED
GFR (ML/MIN/1.73M2
)
1
Normal or minimal kidney damage with
normal GFR
90+
2 Mild decrease in GFR 60-89
3 Moderate decrease in GFR 30-59
4 Severe decrease in GFR 15-29

90. Albumin to Creatinine Ratio
• The urine albumin test or albumin/creatinine ratio (ACR) is
used to screen people with chronic conditions e.g. diabetes and
high blood pressure (hypertension) - increased risk of
developing kidney disease.
• The American Diabetes Association has stated a preference for
the ACR for screening for albuminuria indicating early kidney
disease.
Result Interpretation
< 3 mg/mmol Normal
3.0 - 30.0 mg/mmol Moderately increased
>30.0 mg/mmol Severely increased
Interpretation of ACR:
* Including nephrotic syndrome (urine ACR usually >220 mg/mmol)

91. • ACR is the Albumin Creatinine Ratio - and is the
preferred test for detection of small amounts of
albumin (protein) in the urine.
• A new finding of urine ACR of between 3.0 and 70.0
mg/mmol should be confirmed with a first morning
urine sample to establish consistent finding and exclude
transient elevation due to intercurrent illness.
• If the urine ACR is >70 mg/mmol there is no need to
repeat to confirm the result.
* Regard a confirmed ACR of 3.0 mg/mmol or more as clinically
important proteinuria.

92. Special Tests on urine
1) Porphyrins
Porphyrins require special mention as on the Reagent strip test of
urobilinogen, porphyrins give positive reaction and it has to be
differentiated from urobilinogen.
• The porphyrias are a group of diseases resulting from defects in
the synthesis of heme.
• The Watson-Schwartz test is used to separate causes of a positive
Ehrlich-reacting test and to give an indication of large amounts of
urobilinogen or thepresence of porphobilinogen.

93. Watson-Schwartz test
• Watson Schwartz test is based on solubility differences between
urobilinogen and porphobilinogen. Urobilinogen can be
extracted by chloroform and/or butanol, whereas
porphobilinogen will remain in an aqueous phase.
• A positive result for porphobilinogen in the Watson-Schwartz test
can be further confirmed by the Hoesch test, because the former
may show false-positive results for porphobilinogen as a result of
drugs such as methyldopa.
• Fluorescence screening procedure for porphyrin is another
method using Wood’s lamp to diagnose Porphyrias.

94. Watson- Schwartz Test for
Toconfirm:Hoesch
test

95. 2) Melanin
• No simple specific test is available for melanuria
• Tests based on nonspecific colour reactions produced with ferric
chloride, Ehrlich’s aldehyde reagent, and nitroferricyanide can be
performed.
• A column cation-exchange chromatographic method allows
detection of melanin metabolites in urine.
3) 5-Hydroxyindoleacetic Acid
• It is a metabolite of Serotonin (5-Hydroxytryptophan) found in
urine.
• The metabolite can be analyzed directly by using High
Performance Liquid Chromatography (HPLC).

96. 4) Bence Jones Proteinuria
• Bence Jones proteinuria is associated with multiple myeloma,
macroglobulinemia, and malignant lymphomas.
• Electrophoresis and immunofixation electrophoresis methods are the
best detection and quantification methods, along with the immunoassay
measurement of free light chains
5) Microalbuminuria
• Microalbuminuria is the presence of albumin in urine above the normal
level but below the detectable range of conventional urine dipstick
methods. (Range: 20-200 mg/L – Ref: Todd and Stanford)
• Immunologic test systems and dye-binding chemical test strips are
available for accurate diagnosis
• Important for monitoring Early diabetic nephropathy

97. 6) Aminoacidurias
• Excretion of one or more amino acids in the urine may be due to a
block in a major metabolic pathway (overflow type) or a
deficiency in renal tubular function (renal type).
a) Phenylketonuria: Phenylketonuria is an autosomal recessive
inherited disorder in which there is absence of the enzyme
phenylalanine hydroxylase.
b) Alkaptonuria: The enzyme homogentisic acid oxidase is
deficient, and homogentisic acid is excreted in urine in large
quantities.
Screening Phenistix reagent strips
Confirmatory Ion-exchange high performance liquid
chromatography
Screening Ferric chloride and silver nitrate tests
Confirmatory Paper or thin-layer chromatography; capillary
electrophoresis

98. c) Tyrosinuria:
• Type I hereditary tyrosinemia (tyrosinosis) is an autosomal recessive
disorder characterized by defects in fumarylacetoacetate hydrolase and
maleylacetoacetate hydrolase.
• Type II tyrosinemia (Richner-Hanhart syndrome) is an autosomal
recessive inherited deficiency of tyrosine aminotransferase.
d) Maple Syrup Urine Disease (MSUD):
• Deficient decarboxylases and other enzymes are thought to prevent the
conversion of keto-acids to fatty acids.
Screening Nitrosonaphthol test for tyrosine
Confirmatory Chromatography or quantitative serum assay of
tyrosine
Screening Dinitrophenylhydrazine screening test
Confirmatory Gas or thin-layer chromatographic analysis and nuclear
magnetic resonance spectroscopy

99. Others
24- hour Urinary electrolytes measurement:
• It includes Sodium, Potassium, Sodium:Potassium ratio, Calcium,
etc.
Therapeutic drug monitoring:
• Alcohol, Amphetamines, Benzodiazepines, etc can be monitored
through urine.

100. Recreational drug screening
• To determine if the subject has been using the drug or drugs in
question
• Various occasions when drug testing is done:
1. Pre-employment
2. College/ Professional athletic drug testing
3. Post accident
4. Safety related
• Employers may use a standard five-panel test of "street
drugs" OR may use a nine or ten- panel test according to their
policies.

101. Commonly screened recreational drug-list:
• Opium
• LSD
• Barbiturates/ Benzodiazepines
• Amphetamines
• Ecstasy
• Cocaine
• Psychedelic Mushrooms
• Solvents