1. Assistant Professor of Biochemistry
Examination of Urine
Dr/Mohamed Mahmoud Nour Eldein
PhD Biochemistry
Assistant Professor of Biochemistry
Faculty of Medicine
Umm-ALOura University

2. Urinary System

3. Introduction
Urine is formed in the kidneys, is a product of ultrafiltration of plasma by the renal glomeruli which is a network of arteriolar capillaries, each glomeruli is surrounded by Bowman’s capsule (a double epithelial sac) like a rounded funnel which leads to the tube.

4. Purpose Urine contains important metabolic information
Urine is cheap, simple, & readily available
General evaluation of health
Diagnosis of disease or disorders of the kidneys or urinary tract
Diagnosis of other systemic disease that affect kidney function
Monitoring of patients with diabetes
Screening for drug abuse (eg. Sulfonamide or aminoglycosides)

5. Collection of urine specimens
The first voided morning urine (most concentrated) - qualitative
Random urine (routine)
24hrs sample- quantitative
Mid-stream clean catch (MSCC) (for urine culture)- UTI
Post prandial sample-D.M
Attention
Need to be examined within 1 hour

6. Clean Catch

8. 24 hour urine sample For quantitative estimation of proteins
For estimation of vanillyl mandelic acid, 5-hydroxyindole acetic acid, metanephrines
For detection of AFB in urine
For detection of microalbuminuria

9. Types of Analysis 1- Macroscopic Examination
Physical characteristics : color, odor, turbidity, volume, & specific gravity
Chemical Analysis (Urine Dipstick) : pH, glucose, protein, ketones, pus (WBC’s & bacteria), RBC’s, hemoglobin, bile
2- Microscopic Examination of urine sediment: crystals, cells, etc.

10. Physical examination Volume Color Odour Reaction or urinary pH
Specific gravity

11. Urinary volume
The average daily urine output mL ( L)
The normal daily range of urine output mL ( L)
Polyuria- >2000ml
Oliguria- <400ml
Anuria-complete cessation of urine(<200ml)
Nocturia-excretion of urine by an adult of >500ml with a specific gravity of <1.018 at night (characteristic of chronic glomerulonephritis)

12. Causes of polyuria > 2000ml
Diabetes mellitus
Diabetes insipidus
Polycystic kidney
Chronic renal failure
Diuretics
Intravenous saline/glucose

13. Oliguria <400ml Dehydration-vomiting, diarrhea, excessive sweating
Renal ischemia
Acute tubular necrosis
Obstruction to the urinary tract
Acute renal failure

14. Urine Color and Clarity
Urine color and clarity can indicate what substances may be present in urine.
Confirmation of suspected substances is obtained during the chemical and microsopic examination.

15. Urine Color
Normal urine color ranges from pale yellow to deep amber — the result of a pigment called urochrome
Most changes in urine color are harmless and temporary and may be due to:
Certain foods – beets may turn urine red
Dyes in foods/drinks
Supplements – vitamins
Prescription drugs

16. Abnormal colors of urine & possible causes
Unusual urine color can indicate an infection or serious illness .
Colourless- dilution, diabetes mellitus, diabetes insipidus, diuretics
Milky- genitourinary tract infection
Orange-fever, excessive sweating, bilirubin
Red-beetroot ingestion,haematuria
Brown/ black- alkaptunuria, melanin
Green - bile, Pseudomonas bacteria

17. Examples of Urine Color

18. Urine Clarity Urine clarity refers to how clear the urine is.
Terms used: clear, transparent, slightly cloudy, cloudy, or turbid.
“Normal” urine can be clear or cloudy.
The clarity of the urine is not as important as the substance that is causing the urine to be cloudy.
Turbidity - “cloudiness” due to
particulate matter
suspended in urine

19. Urine Clarity
Substances that cause cloudiness but that are not considered unhealthy include:
mucous,
sperm and prostatic fluid,
cells from the skin,
normal urine crystals, and
contaminants (like body lotions and powders).
Other substances that can make urine cloudy (such as red blood cells, white blood cells, or bacteria) indicate a condition that requires attention.

20. Examples of Urine Clarity

21. Odour Normal= aromatic due to the volatile fatty acids
Standing (“old”) urine takes on an ammonia odor due to urea-splitting bacterial
Foul, offensive: Old specimen, pus or inflammation
Sweet: Glucose
Fruity: Ketones

22. Chemical Analysis

23. Urine dipsticks (Reagent Strips)
Urine dipstick are plastic strips on which are attached to a series of chemically impregnated absorbent pads, each pad contain certain chemicals that react with substance in the urine producing a color change in pad, this color change is compared with a series of known standards.

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

25. Reagent Strips

26. Procedure Testing Reagent strips are used only once and discarded.
Perform within 1 hour after collection
Allow refrigerated specimens to return to room temperature.
Dip strip briefly, but completely into well mixed, room temperature urine sample.
Withdraw strip.
Blot briefly on its side.
Keep the strip flat, read results at the appropriate times by comparing the color to the appropriate color on the chart provided.

27. Procedure
Instruments are available which detect color changes electronically and prints out results

28. Handling and Storage of Strips
Keep strips in original container
Do not touch reagent pad areas
Reagents and strips must be stored properly to retain activity
Protect from moisture and volatile fumes
Stored at room temperature
Use before expiration date

29. Sources of Error
Timing - Failure to observe color changes at appropriate time intervals may cause inaccurate results.
Lighting - Observe color changes and color charts under good lighting.
QC - Reagent strips should be tested with positive controls on each day of use to ensure proper reactivity.
Sample - Proper collection and storage of urine is necessary to insure preservation of chemical.

30. Sources of Error
Testing cold specimens - would result in a slowing down of reactions; test specimens when fresh or bring them to RT before testing
Inadequate mixing of specimen - could result in false reduced or negative reactions to blood and leukocyte tests; mix specimens well before dipping
Over-dipping of reagent strip - will result in leaching of reagents out of pads; briefly, but completely dip the reagent strip into the urine

31. Horseradish Peroxidase
The Urine Dipstick:
Glucose
Chemical Principle
Negative
Trace (100 mg/dL)
+ (250 mg/dL)
++ (500 mg/dL)
+++ (1000 mg/dL)
++++ (2000+ mg/dL)
Glucose Oxidase
Glucose + 2 H2O + O2 --->
Gluconic Acid + 2 H2O2
Horseradish Peroxidase
3 H2O2 + KI ---> KIO3 + 3 H2O
Read at 30 seconds
RR: Negative

32. Uses and Limitations of Urine Glucose Detection
Significance
Diabetes mellitus.
Renal glycosuria.
Limitations
Interference: reducing agents, ketones.
Only measures glucose and not other sugars.
Renal threshold must be passed in order for glucose to spill into the urine.
Other Tests
CuSO4 test for reducing sugars.

33. Urinalysis Glucose Result
Urine versus Blood Glucose ++ +
Urinalysis Glucose Result
trace
Negative
200
400
600
800
1000
Blood Glucose (mg/dL)

34. The Urine Dipstick: Bilirrubin Negative Chemical Principle + (weak)
++ (moderate)
+++ (strong)
Chemical Principle
Acidic
Bilirubin + Diazo salt > Azobilirubin
Read at 30 seconds
RR: Negative

35. Bilirubin Bilirubin is a byproduct of the breakdown of hemoglobin.
Normally contains no bilirubin.
Presence may be an indication of liver disease, bile duct obstruction or hepatitis.
Since the bilirubin in samples is sensitive to light, exposure of the urine samples to light for a long period of time may result in a false negative test result.

36. Ketones
Ketones are excreted when the body metabolizes fats incompletely (ketonuria)

37. Acetoacetic Acid + Nitroprusside ------> Colored Complex
The Urine Dipstick:
Ketones
Negative
Trace (5 mg/dL)
+ (15 mg/dL)
++ (40 mg/dL)
+++ (80 mg/dL)
++++ (160+ mg/dL)
Chemical Principle
Acetoacetic Acid + Nitroprusside
------> Colored Complex
Read at 40 seconds
RR: Negative

38. Uses and Limitations of Urine Ketone Detection
Significance
- Diabetic ketoacidosis
- Prolonged fasting
Limitations
- Interference: expired reagents (degradation with exposure to moisture in air)
- Only measures acetoacetate not other ketone bodies (such as in rebound ketosis).
Other Tests
- Ketostix (more sensitive tablet version of same assay)
- Serum glucose measurement to confirm DKA

39. Specific gravity
Depends on the concentration of various solutes in the urine.
Measured by-urinometer
- refractometer
- dipsticks
Specific gravity reflects kidney's ability to concentrate.
Want concentrated urine for accurate testing, best is first morning sample.
Low – specimen not concentrated, kidney disease.
High – first morning, certain drugs

40. Urinometer Take 2/3 of urinometer container with urine
Allow the urinometer to float into the urine
Read the graduation at the lowest level of urinary meniscus
Correction of temperature & albumin is a must.
Urinometer is calibrated at 15or 200c
So for every 3oc increase/decrease add/subtract 0.001
For 1gm/dl of albumin add0.001

42. The Urine Dipstick: Specific Gravity 1.000 Chemical Principle 1.005
1.010
1.015
1.020
1.025
1.030
Chemical Principle
X+ + Polymethyl vinyl ether / maleic anhydride
>
X+-Polymethyl vinyl ether / maleic anhydride + H+
H+ interacts with a Bromthymol Blue indicator to
form a colored complex.
Read up to 2 minutes
RR:

43. Uses and Limitations of Urine Specific Gravity
Significance
- Diabetes insipidus
Limitations
- Interference: alkaline urine
- Does not measure non-ionized solutes (e.g. glucose)
Other Tests
- Refractometry
- Hydrometer
- Osmolality measurement (typically used with water deprivation test)

44. High specific gravity(hyperosthenuria)
Normal
Causes
All causes of oliguria
Glycosuria

45. Low specific gravity(hyposthenuria)
All causes of polyuria except glycosuria
Fixed specific gravity (isosthenuria)=1.010
Seen in chronic renal disease when kidney has lost the ability to concentrate or dilute

46. Blood
Presence of blood may indicate infection, trauma to the urinary tract or bleeding in the kidneys.
False positive readings most often due to contamination with menstrual blood.

47. The Urine Dipstick: Blood Negative Trace (non-hemolyzed)
Chemical Principle
Trace (non-hemolyzed)
Lysing agent to lyse red blood cells
Moderate (non-hemolyzed)
Diisopropylbenzene dihydroperoxide +
Tetramethylbenzidine
> Colored Complex
Trace (hemolyzed)
Heme
+ (weak)
++ (moderate)
Read at 60 seconds
RR: Negative
Analytic Sensitivity: 10 RBCs
+++ (strong)

48. Uses and Limitations of Urine Blood Detection
Significance
- Hematuria (nephritis, trauma, etc)
- Hemoglobinuria (hemolysis, etc)
- Myoglobinuria (rhabdomyolysis, etc)
Limitations
- Interference: reducing agents, microbial peroxidases
- Cannot distinguish between the above disease processes
Other Tests
- Urine microscopic examination
- Urine cytology

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

50. Methyl Red (at high concentration; low pH) and
The Urine Dipstick: pH
5.0
6.0
6.5
7.0
7.5
8.0
8.5
Chemical Principle
H+ interacts with:
Methyl Red (at high concentration; low pH) and
Bromthymol Blue (at low concentration; high pH), to form a colored complexes (dual indicator system)
Read up to 2 minutes
R.R.:

51. Acidic urine Ketosis-diabetes, starvation, fever Systemic acidosis
UTI- E.coli
Acidification therapy

52. Alkaline urine Strict vegetarian Systemic alkalosis UTI- Proteus
Alkalization therapy

53. Uses and Limitations of Urine pH Detection
Significance
- Acidic (less than 4.5): metabolic acidosis, high-protein diet
- Alkaline (greater than 8.0): renal tubular acidosis (>5.5)
Limitations
- Interference: bacterial overgrowth (alkaline or acidic),
“run over effect” effect of protein pad on pH indicator pad
Other Tests
- Titrable acidity
- Blood gases to determine acid-base status

54. pH Run Over Effect Glucose Bilirubin Ketones
Specific Gravity
Blood pH
Protein
Urobilinogen
Nitrite
Leukocyte Esterase
Buffers from the protein area of the strip (pH 3.0) spill over to the pH area of the strip and make the pH of the sample appear more acidic than it really is.

55. Protein
Presence of protein (proteinuria) is an important indicator of renal disease.
False negatives can occur in alkaline or dilute urine or when primary protein is not albumin.

56. The Urine Dipstick: Protein Chemical Principle
“Protein Error of Indicators Method”
Negative
Trace
+ (30 mg/dL)
++ (100 mg/dL)
+++ (300 mg/dL)
++++ (2000 mg/dL) Pr H
Tetrabromphenol Blue
(buffered to pH 3.0) H+ Pr Pr Pr Pr Pr Pr
Read at 60 seconds
RR: Negative

57. Causes of Proteinuria Functional Renal Pre-Renal Post-Renal
- Severe muscular exertion - Glomerulonephritis
- Pregnancy - Nephrotic syndrome
- Orthostatic proteinuria - Renal tumor or infection
Pre-Renal Post-Renal
- Fever - Cystitis
- Renal hypoxia - Urethritis or prostatitis
- Hypertension - Contamination with vaginal secretions

58. Uses and Limitations of Urine Protein Detection
Significance
- Proteinuria and the nephrotic syndrome.
Limitations
- Interference: highly alkaline urine.
- Much more sensitive to albumin than other proteins
(e.g., immunoglobulin light chains).
Other Tests
- Sulfosalicylic acid (SSA) turbidity test.
- Urine protein electrophoresis (UPEP)
- Bence Jones protein

59. Urobilinogen
Urobilinogen is a degradation product of bilirubin formed by intestinal bacteria.
It may be increased in hepatic disease or hemolytic disease

60. Urobilinogen + Diethylaminobenzaldehyde -------> Colored Complex
The Urine Dipstick:
Urobilinogen
0.2 mg/dL
1 mg/dL
2 mg/dL
4 mg/dL
8 mg/dL
Chemical Principle
Urobilinogen + Diethylaminobenzaldehyde
> Colored Complex
(Ehrlich’s Reagent)
Read at 60 seconds
RR: mg/dL

61. Uses and Limitations of Urobilinogen Detection
Significance
- High: increased hepatic processing of bilirubin
- Low: bile obstruction
Limitations
- Interference: prolonged exposure of specimen to oxygen (urobilinogen ---> urobilin)
- Cannot detect low levels of urobilinogen
Other Tests
- Serum total and direct bilirubin

62. Nitrite
Nitrite formed by gram negative bacteria converting urinary nitrate to nitrite

63. The Urine Dipstick: Nitrite Chemical Principle Negative Positive
Acidic
Negative
Positive
Nitrite + p-arsenilic acid > Diazo compound
Diazo compound + Tetrahydrobenzoquinolinol
> Colored Complex
Read at 60 seconds
RR: Negative

64. Uses and Limitations of Nitrite Detection
Significance
- Gram negative bacteriuria
Limitations
- Interference: bacterial overgrowth
- Only able to detect bacteria that reduce nitrate to nitrite
Other Tests
- Correlate with leukocyte esterase and
- Urine microscopic examination (bacteria)
- Urine culture

65. Leukocytes Leukocytes (white blood cells) usually indicate infection.
Leucocyte esterase activity is due to presence of WBCs in urine while nitrites strongly suggest bacteriuria.

66. The Urine Dipstick: Leukocyte Esterase Chemical Principle
Derivatized pyrrole amino acid ester
> 3-hydroxy-5-phenyl pyrrole
Negative
Trace
+ (weak)
++ (moderate)
+++ (strong)
Esterases
3-hydroxy-5-phenyl pyrrole + diazo salt
> Colored Complex
Read at 2 minutes
RR: Negative
Analytic Sensitivity: 3-5 WBCs

67. Significance Limitations Other Tests
Uses and Limitations of Leukocyte Esterase Detection
Significance
- Pyuria
- Acute inflammation
- Renal calculus
Limitations
- Interference: oxidizing agents, menstrual contamination
Other Tests
- Urine microscopic examination (WBCs and bacteria)
- Urine culture

68. Normal Values
Negative results for glucose, ketones, bilirubin, nitrites, leukocyte esterase and blood.
Protein negative or trace. pH
Urobilinogen Ehrlich units

69. Microscopic examination
Microscopic urinalysis is done simply pouring the urine sample into a test tube and centrifuging it (spinning it down in a machine) for a few minutes. The top liquid part (the supernatant) is discarded. The solid part left in the bottom of the test tube (the urine sediment) is mixed with the remaining drop of urine in the test tube and one drop is analyzed under a microscope

70. Microscopic Examination
Abnormal Findings
Per High Power Field (HPF) (400x)
> 3 erythrocytes
> 5 leukocytes
> 2 renal tubular cells
> 10 bacteria
Per Low Power Field (LPF) (200x)
> 3 hyaline casts or > 1 granular cast
> 10 squamous cells (indicative of contaminated specimen)
Any other cast (RBCs, WBCs)
Presence of:
Fungal hyphae or yeast, parasite, viral inclusions
Pathological crystals (cystine, leucine, tyrosine)
Large number of uric acid or calcium oxalate crystals

71. Microscopic Examination
Cells
Erythrocytes
- “Dysmorphic” vs. “normal” (> 10 per HPF)
Leukocytes
- Neutrophils (glitter cells) More than 1 per 3 HPF
- Eosinophils Hansel test (special stain)
Epithelial Cells
- Squamous cells Indicate level of contamination
- Renal tubular epithelial cells Few are normal
- Transitional epithelial cells Few are normal
- Oval fat bodies Abnormal, indicate Nephrosis

72. Microscopic Examination
RBCs

73. Microscopic Examination
RBCs

74. Microscopic Examination
WBCs

75. Microscopic Examination
Squamous Cells

76. Tubular Epithelial Cells
Microscopic Examination
Tubular Epithelial Cells

77. Microscopic Examination
Transitional Cells

78. Microscopic Examination
Transitional Cells

79. Microscopic Examination
Oval Fat Body

80. Microscopic Examination
LE Cell

81. Microscopic Examination
Bacteria & Yeasts
Bacteria
- Bacteriuria More than 10 per HPF
Yeasts
- Candidiasis Most likely a contaminant
but should correlate with
clinical picture.
Viruses
- CMV inclusions Probable viral cystitis.

82. Microscopic Examination
Bacteria

83. Microscopic Examination
Yeasts

84. Microscopic Examination
Yeasts

85. Microscopic Examination
Cytomegalovirus

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

87. Microscopic Examination
Casts

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

89. Microscopic Examination
Casts
Erythrocyte Casts: Glomerular diseases
Leukocyte Casts: Pyuria, glomerular disease
Degenerating Casts:
- Granular casts Nonspecific (Tamm-Horsfall protein)
- Hyaline casts Nonspecific (Tamm-Horsfall protein)
- Waxy casts Nonspecific
- Fatty casts Nephrotic syndrome
(oval fat body casts)

91. Red cell casts
The presence of red blood cells within the cast is always pathologic, and is strongly indicative of glomerular damage.
They are usually associated with nephritic syndromes.

92. Microscopic Examination
RBCs Cast - Histology

93. Microscopic Examination
RBCs Cast

94. Microscopic Examination
RBCs Cast - Histology

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

97. Microscopic Examination
WBCs Cast

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

99. Microscopic Examination
Tubular Epith. Cast

100. Microscopic Examination
Tubular Epith. Cast

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

102. Microscopic Examination
Granular Cast

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

104. Microscopic Examination
Hyaline Cast

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

106. Waxy casts

107. Microscopic Examination
Waxy Cast

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

109. Fatty casts

110. Microscopic Examination
Fatty Cast

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

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

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

114. crystals

115. Calcium Oxalate Crystals
Microscopic Examination
Calcium Oxalate Crystals

116. Calcium Oxalate Crystals
Microscopic Examination
Calcium Oxalate Crystals
Dumbbell Shape

117. Triple Phosphate Crystals
Microscopic Examination
Triple Phosphate Crystals

118. Microscopic Examination
Urate Crystals

119. Microscopic Examination
Cystine Crystals