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Examination of Urine

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Title: Examination of Urine


1
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 Bowmans 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
7

8
24 hour urine sample
  1. For quantitative estimation of proteins
  2. For estimation of vanillyl mandelic acid,
    5-hydroxyindole acetic acid, metanephrines
  3. For detection of AFB in urine
  4. 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 (WBCs bacteria), RBCs,
    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 1200 - 1500 mL
    (1.2 - 1.5 L)
  • The normal daily range of urine output 600 -
    2000 mL (0.6 - 2.0 L)
  • Polyuria- gt2000ml
  • Oliguria- lt400ml
  • Anuria-complete cessation of urine(lt200ml)
  • Nocturia-excretion of urine by an adult of gt500ml
    with a specific gravity of lt1.018 at night
    (characteristic of chronic glomerulonephritis)

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

13
Oliguria lt400ml
  • 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
25
Reagent Strips
26
Procedure
  • Reagent strips are used only once and discarded.
  • Testing
  • 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
  • Handling and Storage
  • 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
The Urine Dipstick
Glucose
Chemical Principle
Glucose Oxidase
Glucose 2 H2O O2 ---gt Gluconic Acid 2 H2O2
Horseradish Peroxidase
3 H2O2 KI ---gt 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
Urine versus Blood Glucose


Urinalysis Glucose Result
trace
Negative
400
600
800
1000
200
Blood Glucose (mg/dL)
34
The Urine Dipstick
Bilirrubin
Chemical Principle
Acidic
Bilirubin Diazo salt ---------gt 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
The Urine Dipstick
Ketones
Chemical Principle
Acetoacetic Acid Nitroprusside ------gt 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

41

42
The Urine Dipstick
Specific Gravity
Chemical Principle
X Polymethyl vinyl ether / maleic
anhydride ---------------gt 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 1.003-1.035
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-1.016-1.022
  • 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
Chemical Principle
Trace (non-hemolyzed)
Lysing agent to lyse red blood cells
Moderate (non-hemolyzed)
Diisopropylbenzene dihydroperoxide
Tetramethylbenzidine ------------gt Colored
Complex
Trace (hemolyzed)
Heme
(weak)
Read at 60 seconds RR Negative Analytic
Sensitivity 10 RBCs
(moderate)
(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
The Urine Dipstick
pH
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. 4.5-8.0
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 (gt5.5)
  • 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
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
Tetrabromphenol Blue (buffered to pH 3.0)
Pr
Pr
Pr
Pr
Pr
Pr
Read at 60 seconds RR Negative
57
Causes of Proteinuria
  • Functional 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
The Urine Dipstick
Urobilinogen
Chemical Principle
Urobilinogen Diethylaminobenzaldehyde -------gt
Colored Complex
(Ehrlichs Reagent)
Read at 60 seconds RR 0.02-1.0 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 ---gt 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
Acidic
Nitrite p-arsenilic acid -------gt Diazo compound
Diazo compound Tetrahydrobenzoquinolinol -------
---gt 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
------------gt 3-hydroxy-5-phenyl pyrrole
Esterases
3-hydroxy-5-phenyl pyrrole diazo
salt -------------gt Colored Complex
Read at 2 minutes RR Negative Analytic
Sensitivity 3-5 WBCs
67
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 5.5-8.0
  • Urobilinogen 0.2-1.0 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)
  • gt 3 erythrocytes
  • gt 5 leukocytes
  • gt 2 renal tubular cells
  • gt 10 bacteria
  • Per Low Power Field (LPF) (200x)
  • gt 3 hyaline casts or gt 1 granular cast
  • gt 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 (gt 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
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)

90

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

96
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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 RBCs,
    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
Microscopic Examination
Calcium Oxalate Crystals
116
Microscopic Examination
Calcium Oxalate Crystals
Dumbbell Shape
117
Microscopic Examination
Triple Phosphate Crystals
118
Microscopic Examination
Urate Crystals
119
Microscopic Examination
Cystine Crystals
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