Professional Documents
Culture Documents
09/28/22 2
Chapter outline
Determination of Urinary Ph
Determination of Urinary sugar
Determination of Urinary keton bodies
Determination of Urinary protein
Determination of Urinary hemoglobin
Determination of Urinary Bilirubin
Determination of Urinary urobilinogen
Determination of Urinary urobilin
Determination of Urinary Porphybilinogen
Determination of Urinary lecuocyte estrase
Determination of Urinary Nitrite
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Chapter outline cont’d
09/28/22 4
Introduction
Chemical analysis of urine deals with those procedures
or examinations which are accomplished by means of
chemical reactions. .
Chemical test are done for a quick screening of urine for
certain substances which have a diagnostic significance.
In this category of tests, abnormal or pathological
dissolved constituents of urine (chemicals) performed in
routine urinalysis
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Introduction Cont…
09/28/22 6
Introduction Cont…
Urine composition depends greatly on how much and what
specific waste material is to be excreted.
Urea, creatinine, uric acid, ammonium salts, chlorides, sulphates
and phosphates of sodium, potassium, calcium and magnesiumn are
the normal composition of urine.
Glucose, protein, ketone bodies, bilirubin , bile salts etc. are the
abnormal constituents of urine.
Normally these substances do not appear in the urine in
detectable amount .
So their appearance in the urine shows the pathological condition.
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Chemical analysis
Chemical analysis of urine can be:
Screening test: tells the presence or absence of the abnormal
components of urine
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PH
Along with the lungs, the kidneys are the major regulators
of the acid-base content in the body.
They do this through the secretion of hydrogen in the
form of ammonium ions, hydrogen phosphate, and weak
organic acids, and by the reabsorption of bicarbonate
from the filtrate in the convoluted tubules
A healthy individual usually produces a first morning
specimen with a slightly acidic pH of 5.0 to 6.0; a more
alkaline pH is found following meals (alkaline tide).
The pH of normal random samples can range from 4.5 to
8.0.
09/28/22 9
PH cont.
pH is the unit that describes the acidity or alkalinity of a
solution
In chemical terms, acidity refers to the hydronium ion
(H3O+) concentration of a solution and alkalinity refers to
its hydroxyl ion (OH-) concentration.
These concentrations are usually expressed in terms of pH.
Because the kidney is generally working to eliminate excess
acid, the pH of the urine is normally between 5 and 6.
The kidney is capable of producing urine ranging in pH
from 4.5-8.0.
Although the kidney is essential in controlling the pH of
blood and extracellular fluid, measurement of urinary pH are
not necessarily used to obtain information about this role.
09/28/22 10
Reasons for measurement of urinary pH
Alkalinity of freshly voided urine, indicate a urinary
tract infection.
The urinary pH helps in the identification of crystals
of certain chemical compounds
Persistently acidic urine in diabetic acidosis
resulting from an accumulation of ketone bodies.
To control management of kidney infections, in
cases of renal calculi (stones) and during the
administration of certain drugs.
09/28/22 11
Method of measuring urinary pH
1. Litmus paper, 3. Nitrazine paper
2. Reagent strip and 4. Glass electrode (pH meter)
Litmus paper
In this technique pH measurement takes place by using
blue, and red litmus paper.
Procedure
Collect a freshly voided well mixed urine
litmus paper.
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Interpretation:
If
blue colors of paper changes to red, it indicates
the acidity of the urine. If the red litmus paper
change to blue, it indicates that the urine is alkaline.
Nitrazine paper
The paper is impregnated with sodium dinitro
phenolazo – naphthal disulphonate chemical.
a paper that changes its color from yellow (for acidic
urine) to blue (for alkaline urine).
Unlike litmus paper, the color change is matched with
reference color chart.
09/28/22 13
Procedure
Tear small nitrazine paper
Dip the paper in freshly voided urine sample and
remove immediately
Compare the color change with that of reference color
chart.
Record the value of color change form reference color
chart.
Note: Most nitrazine paper is manufactured with color
charts ranging from pH 3 or 4 to pH 9. However,
nitrazine paper has an accurate range of only 4.5-7.5.
09/28/22 14
Reagent Strip Test
Principle
Utilize a methyl red and bromthymol blue double
indicator system that measures urine pH from 5 to 9.
The Methyl red is used to indicate a pH range from
4.4 to 6.2 with a color change from red to yellow
Bromthymol blue indicates a pH change from 6.0 to
7.6 as seen by a color change from yellow to blue.
09/28/22 15
Chemical Exam
When the test strip is
dipped in urine the
reagents are activated
and a chemical
reaction occurs.
The chemical reaction
results in a specific
color change.
09/28/22 16
Procedure
Dip the test – strip in the urine specimen. Remove the test-strip
immediately and let the excess urine drain off on a paper towel, or
tap the edge of the strip.
Read the color change
Report the result according to the color chart provided by
manufacturer.
Always read the test strip in good white light and ignore color
developing on the test area after the period specified as the
reading time of the test.
Be careful not to wet the reagent strip excessively. So that the
acid buffer from the protein area runs into the pH area, causing an
orange discoloration.
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Elevated pH (alkaline)
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General Precaution for reagent strips
Store strips in a cool, dry place to prevent
deterioration
Do not use strips if they are discolored
Do not touch the test areas
Keep the test areas away from detergents or other
contaminating substances
Dip the test areas into the urine specimen completely,
but briefly to avoid dissolving the reagents out of the
test area.
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General Precaution for reagent
strips …
Remove only the number of reagent strips required
at a particular time, and keep the container tightly
covered at all times to prevent deterioration
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pH meter
Hydrogen ion activity can be
measured with a pH meter
calibrated to this scale of 0-
14 with standard solutions.
The meter measures
potential of hydrogen ion
activity or pH of an aqueous
solution.
09/28/22 24
pH Meter
The pH
measurement
system consists of 2
electrodes:
a measuring
electrode capable of
detecting hydrogen
ions and
the reference
electrode
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pH measurement
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pH measurement
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Clinical Significance
As indicated, one of the functions of renal system is to
regulate pH of blood i.e. keep pH of blood at 7.4 ± 0.05.
This is done by absorption or release of hydrogen ion,
especially at distal convoluted tubules of the nephron,
depending on the pH of blood, i.e. hydrogen ion
absorbed from surrounding blood capillaries of nephron
when pH is acidic (below 7.35), and release from
nephron to the surrounding blood vessels when pH of
blood is alkaline (above 7.45).
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Clinical Significance cont’d…
Persistent alkaline urine (pH > 6)may be caused by:
UTI
Renal failure
Vomiting
Anorexia nervosa
metabolic Alkalosis.
Alkalizing drugs such as streptomycin, kanamycin etc.
Certain vegetables, like citrus fruits.
milk products
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Clinical Significance cont’d…
Persistent acid urine (pH < 6) may be caused by:
Diarrhea
Malabsorption syndromes
Diabetic ketoacidosis
Dehydration
Fever
Starvation
And also certain drugs such as – Phenacetin
high protein diet may also result in acidic urine,
but this is not a pathological condition.
09/28/22 30
Causes of Acid and Alkaline Urine
09/28/22 31
Determination of Glucose
Glucose is the sugar most commonly found in the urine,
although other sugars , such as lactose, fructose, galactose, and
pentose, may be found under certain condition.
The presence of detectable amount of glucose in the urine is
known as glycosuria.
Normally almost all the glucose, which passes from the blood into
the glomerular filtrate, is reabsorbed back into the circulation by
the kidney tubules( proximal convoluted tubules ).
Usually less than 15 - 20 mg/dl (0.8 mmol) is excreted in the urine.
But this amount cannot be detected by the routine laboratory
tests.
09/28/22 32
Glucose cont’d…
The blood glucose concentration normally lies between 65 and
110 mg/dl.
After a meal it may increase to 120 - 160 mg/dl.
If the blood glucose concentration becomes too high (usually
greater than 170 - 180 mg/dl), the excess glucose will not be
reabsorbed into the blood and glucose start appearing in urine.
The lowest blood glucose concentration that will result in
glycosuria is termed as the renal threshold.
The most common condition in which the renal threshold for
glucose exceeds is diabetes mellitus
09/28/22 33
Causes of Glycosuria
Physiological
Pathological
Physiological
a. After large ingestion of carbohydrates
b. Anything that stimulates sympathetic nervous system
c. 15 to 20% cases of pregnancy
d. Renal Glycosuria:
09/28/22 34
Causes of Glycosuria
Pathological Glycosuria
A. Diabetes mellitus
Diabetes Mellitus is a chronic polygenic syndrome
with impaired carbohydrate metabolism.
Diabetes mellitus and its complications (diabetic
keto-acidosis and non ketotic hyperosmolar
syndrome) are the most common disorders of
carbohydrate metabolism.
09/28/22 35
Metabolic changes in diabetes mellitus
09/28/22 36
Causes of Glycosuria
09/28/22 37
Causes of Glycosuria cont’d
The carbohydrate metabolism is impaired due to
deficiency or ineffectiveness of insulin (peripheral
insulin resistance) or decreased insulin/anti-insulin
hormone ratio leading to chronic hyperglycemia and
glycosuria along with secondary changes in
metabolism of protein, lipids, water and electrolytes
with grave consequences, if not treated.
Typically, its symptoms include polydypsia
(excessive thirst), polyuria (increased frequency of
urination), polyphagia (hunger), glucosuria
09/28/22 38
Types of Urinary Glucose Test
Non specific
reduction tests based on the reduction of certain metal
ion by glucose
Specific
Enzymatic tests based on the action of glucose
oxidase on glucose
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Non-Specific Test for Glucose
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Non-Specific Test for Glucose
-Fehling test
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The overall reaction
09/28/22 43
procedure
1. Arrange test tubes (12ml)in a row rack.
2. Take two extra tubes for control runs
negative with water and
positive with 1.0 g/dl of glucose solution.
3. Add eight drops of well-mixed urine
4. Add 5ml of Benedict’s reagent.
5. Mix well and place in a boiling water bath for 5 min
6. Remove and cool to room temperature .
7. A positive : presence of a fine yellow to brick red
8. Graded on the basis of the color of solution.
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Grade results according to the following
criteria:
Fehling’s reagent
Solution A Solution B
Copper sulphate 34.65 g Sodium hydroxide 125 gm
Distilled water 500 ml Sodium potassium
tartarate173 gm
Distilled water 500 ml
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Procedure
09/28/22 47
Advantage of Bendict’s Test over Fehling’s test
09/28/22 48
Clinitest Tablet Tests
Principle:
- The same as that of Benedict's Qualitative Test.
09/28/22 49
Clinitest Tablet Tests have;
Sensitivity
Clinitest reagent tablets will detect as little as
250mg of sugar in 100ml of urine.
09/28/22 50
Procedure for Five drop method
Follow the direction supplied with the clintest tablets
Place 5 drops of urine in a test tube and add 10 drops of
distilled water
Add one clintest tablet by easing it in to the tube with out
touching it. It contains strong alkali.
Watch while boiling takes place, but don’t shake or touch the
bottom of tube
Wait 15 seconds after boiling stops, then shake the tube gently
and compare the color of the solution with the color scale
Grade the results as negative, trace, 1+, 2+, 3+, or 4+. The
results correspond to the following concentrations (mg/dl):
trace, 250mg/dl; 1+, 500mg/dl; 2+, 750mg/dl: 3+, 1000mg/dl:
4+, 2000mg/dl.
09/28/22 51
Specific (enzymatic) test for glucose
09/28/22 52
Specific (enzymatic) test for glucose
Tes tape
Diastistix
09/28/22 53
Principle of Clinistix
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Clinistix Reagent Strip Test
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Chemical Reaction Chart
09/28/22 56
Chemical Exam
When the test strip is
dipped in urine the
reagents are activated
and a chemical
reaction occurs.
The chemical reaction
results in a specific
color change.
09/28/22 57
Positive Glucose
09/28/22 58
Procedure
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Test-Tape for Glucose
Principle
Tes-Tape is a screening test
specific for glucose.
The principle is the same to clinistix
Differ in the oxidation -reduction indicator employed, and the
material the reagents are impregnated on.
In Tes-Tape the reagents are impregnated on a tear strip of
special paper, and the indicator is yellow in its reduced form
and green to blue in its oxidized form.
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Procedure
Follow the manufacture direction:
1. Tear off approximately 1 and 1/2 inch.
2. Dip part of the tape into the urine specimen; remove it
immediately.
3. Wait for 30 seconds; then observe the appearance of any
green color.
4. Record the result as positive or negative.
If the test area remains yellow after 30 seconds, the
result is negative.
If any green color is present at this time, the result is
positive.
09/28/22 61
Diastix Reagent Strip for Glucose
Diastix is a specific test for glucose based on the use
of glucose oxidase,
The chemical reaction is the same as clinistix
the difference being the chromogen system used to
indicate the presence of glucose.
The reagent area contains glucose oxidase,
peroxidase, a blue background dye, and potassium
iodide as the chromogen.
09/28/22 62
Procedure
09/28/22 63
Rubner’s Test
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Fructose
09/28/22 65
Seliwanoff’s Test for Fructose
09/28/22 66
Procedure for Seliwanoff’s Test
Add a few drops of urine (about 0.5 ml) to about 5 ml
of the reagent in a test tube
Boil.
Interpretation
Fructose gives a red color within half a minute.
The test is sensitive to 5.5 mmole/l (100 mg/100
ml) if glucose is absent, but if glucose in present it
is less sensitive and in addition large amount of
glucose can give a similar color.
09/28/22 67
Clinical Significance
Normally, urine doesn't contain a sufficient amount
of sugar to react with any of the popular enzyme
or reducing tests.
When sugar appears in the urine, it shows the
abnormality caused by disease diabetes mellitus.
Test for urine sugar is used in screening to detect
diabetic mellitus, confirming diagnosis of D. mellitus,
and monitoring the effectiveness of diabetic therapy.
09/28/22 68
Ketones
Ketone bodies, also called Ketones,
are a group of three related substances
Acetone
acetoacetate (acetoacetic acid) and
-hydroxybutyrate ( -hydroxybutyric acid).
Formed in liver from aceto acitate (oxidation product of
amino acid, fatty acid)
Used as energy source for extra hepatic tissue like brain
09/28/22 69
Ketone bodies an alternative fuel for cells.
Liver mitochondria have the capacity to divert any excess acetyl
CoA derived from fatty acid or pyruvate oxidation into ketone
bodies.
The compounds categorized as ketone bodies are acetoacetate,
3- hydroxybutyrate and acetone (a volatile non-metabolizable
side product).
They are transported in the blood to peripheral tissues, where
they can be reconverted to acetyl CoA and oxidized by the TCA
cycle.
Ketone bodies are soluble in aqueous solution, and therefore, do
not need to be incorporated in lipoproteins or carried by albumin
as do other lipids.
They are produced in the liver during periods when the amount
of acetyl CoA present exceeds the oxidative capacity of the liver.
09/28/22 70
cont’d
09/28/22 71
cont’d
09/28/22 72
09/28/22 73
09/28/22 74
Tests for ketone bodies in Urine
Rothera’s test
Lang’s test
Acetest tablet test
Acetone powder test
Reagent strip tests (eg. ketostix)
09/28/22 75
Rothera’s test
Principle
Both acetone and acetoacetic acid give a purple color
with alkaline sodium nitroprusside.
This is the general principle for the tests mentioned
above.
Results - Report the test as positive or negative
09/28/22 76
Procedure
09/28/22 77
Ketostix Reagent Strip Test
Principle
Is based on Rothera’s test,
Acetoacetic acid will react with sodium nitroprusside in
a alkaline medium to form a purple color.
If glycin is added, the test is slightly sensitive to acetone.
09/28/22 78
Ketostix Reagent Strip Test
Procedure
Dip test- end of the strip in urine
At 15 seconds compare the color of dipped- end with
the color chart.
Report as indicated by the color chart.
09/28/22 79
Positive urine Ketones
09/28/22 81
Gerhardt’s Test for acetoacetate
Reagent
10% Ferric chloride reagent
Weigh 10 g of ferric chloride and transfer to a 100 ml
volumetric flask.
09/28/22 82
Clinical Significance
When the rate of formation of ketone bodies is greater
than the rate of their use, their levels begin to rise in
the blood, which is called ketonemia, and eventually
in the urine, which is known as ketonuria.
These two conditions are seen most often in cases of
starvation and diabetes mellitus.
Ketone bodies can be seen also in the urine during
prolonged vomiting, severe diarrhea, anesthesia,
severe liver damage, high fat intake and low
carbohydrate diet.
09/28/22 83
Clinical Significance continued
09/28/22 84
Determination of Urinary Protein
Introduction:
Test for urinary protein is one of the most important and
valuable parts of the routine urinalysis.
Albumin is one of the important proteins, which appears
in urine during a pathological condition.
It often occurs as a symptom of renal disease.
Globulins are excreted less frequently.
Bence Jones protein is a specific type of globulin
excreted in multiple myeloma.
09/28/22 85
Causes of Proteinuria
1. Increased permeability of the glomerulus
Normally, the glomerular membrane,in the formation
of urine, is not permeable for protein molecules.
If the glomerular membrane is damaged these large
protein molecules can pass through
2. A decrease in normal reabsorption in the tubules
disease condition which affect reabsorption
09/28/22 86
Types of proteinurea
09/28/22 87
Functional Proteinuria cont’d…
2. Physiological proteinuria:
It is usually less than 0.5 gm/24 hr. which is
associated with fever, exposure to heat or cold,
emotional stress, and later stage of pregnancy.
09/28/22 88
B. Systemic disease or renal pathology
Proteinuria.
1. Pre renal protein urea: not due to primary renal
disease
Fever and a variety of toxic condition
Venous congestion
Renal hypoxia
Hypertension
Myxedema
Bence Jones protein
09/28/22 89
Systemic disease or renal pathology
Proteinuria cont’d…
2.Renal proteinuria: primarily kidney disease
Gomerulonephritis
Nephrotic syndrom, primary and secondary
Destructive parenchymal lesions (tumour,
infection
09/28/22 90
Systemic disease or renal pathology
Proteinuria cont’d…
09/28/22 91
Test for urinary protein
09/28/22 92
Robert's Test
Principle
The principle of this test is based on the precipitation
of protein and formation of white compact ring
using concentrated Nitric acid (HNO3).
09/28/22 93
Procedure of Robert's Test
09/28/22 94
Heller's Test
09/28/22 95
Sulphosalicylic Acid Test
Principle
Sulphosalicylic acid solution (20%) precipitates any
protein in the urine specimen.
It is an anion precipitant that works by the
neutralization of the protein cation.
This method is more sensitive and reliable than the
heat method.
It can detect 5 to 10 mg/dl of protein in urine
specimen
09/28/22 96
Procedure of Sulphosalicylic Acid Test
09/28/22 97
09/28/22 98
Heat and Acetic Acid Test
Procedure
Fill a test tube three-fourth full of clear urine, and gently
heat the upper portion of urine for 2 minutes to boil,
being careful not to shake the tube more than
necessary. The lower portion of urine is not heated
so that it can be used as a control for
comparing.
If turbidity ( a white cloud ) can arise due either of
phosphates, carbonates, or protein.
09/28/22 99
Procedure Cont’d…
09/28/22 100
Heat and Acetic Acid Test cont’d…
Sensitivity
This method is the most sensitive for small amount of
protein and can reliably detect protein concentrations of
2 to 3 mg/dl.
09/28/22 101
Reagent Strip Reactions
09/28/22 102
Reagent Strip Reactions
09/28/22 103
Colorimetric Reagent Strip (Dipstick)
Principle
The Colorimetric reagent strip test is based on the
ability of protein to alter the color of some cid-
base indicators without altering the pH.
When an indicator, such as tetrabromophenol blue
is buffered at pH 3, it is yellow in solutions without
protein.
Tests are more specific.
They require only a drop of urine enough to
moisten the reagent area.
09/28/22 104
Procedure of Reagent Strip
09/28/22 105
Chemical Exam
09/28/22 106
09/28/22 107
Quantitative 24 hour protein Determinations
09/28/22 108
Quantitative 24 hour protein Determinations
09/28/22 109
Quantitative 24 hour protein Determinations
B. Esbach’s Test
1. measure the total volume; then filter some of the
urine.
2. Do qualitative protein test, Robert’s or strip test.
if the urine is +3, made 1:5 dilution.
09/28/22 111
Clinical Significance
Proteinuria: >6-8gm/l.
renal damage cause proteinuria.
A positive protein test for urine may b correlated with
findings of casts.
In cases of renal disease, it is essential that the
diagnosis be made and treatment started as soon as
possible to prevent extensive and permanent renal
damage.
Proteinuria with the presence of leukocytes and bacteria
in the urine will be suggestive of urinary tract infection.
09/28/22 112
Bence - Jones Protein
09/28/22 113
Heat test to screen Bence -Jones Protein
Principle:
Bence Jones protein coagulates when heated to 40 to
600C and re-dissolves partially or wholly on boiling.
Albumin coagulates above 600C and does not re-
dissolve on boiling.
09/28/22 114
Heat test to screen Bence -Jones Protein
Procedure
1. Take 5ml of clear fresh urine into each of three test tubes.
2. Acidify the urine by adding 1 drop of glacial acetic acid to
the first tube, 2 drops to the second tube, and 3 drops to the
third tube.
3. Place the three tubes in a beaker of tap water and insert a
0-100 OC thermometer in one of the tube.
4. Using a gentle flame slowly heat the beaker of water.
5. When the temperature reaches 40OC look for cloudiness
and precipitate in the tubes. Continue to observe until the
temperature reaches 60OC.
09/28/22 115
Clinical significance
09/28/22 116
Determination of Hemoglobin
09/28/22 117
Clinical significance
09/28/22 118
Causes of Intravascular Hemolysis
09/28/22 119
Causes of Intravascular Hemolysis Cont’d…
09/28/22 120
Determination of Hemoglobin
09/28/22 121
Determination of Hemoglobin cont’d…
Principle
Presence of peroxidase activity of the hemoglobin and
hydrogen
peroxide (H2O2) which librates oxygen.
The intensity of the color depends on amount of librated
oxygen.
The amount of librated oxygen depends on the peroxidase
activity of hemoglobin molecule, which intern depends on
the amount of hemoglobin found in the urine.
09/28/22 122
Principle cont’d…
09/28/22 123
Factors that Affect Hemoglobin
Determination
False negative
High specific gravity such as heavy Proteinuria (over
5 g/1). This prevent lysis of RBCs and may reduce
the color reaction.
Low to false negative results are obtained if the urine
contains large amounts of ascorbic acid.
Nitrite delays test reaction.
Formaline used as preservative, fix the cell and
prevent hemolysis.
09/28/22 124
Cont…
False positive
Low specific gravity < 1.010 enhances lysis and
produces color reaction.
Microbial peroxidase produces false the color
reaction.
presence of contaminating oxidizing detergents on
the urine such as bleach.
09/28/22 125
Benzidine Test
Principle
The peroxidase activity of hemoglobin
decomposes hydrogen peroxide and the
liberated active oxygen oxidizes the organic
compound Benzidine.
09/28/22 126
Procedure
(1) Mix equal parts (1mL each) of A and B in a test tube
(15-mL) just before use.
(2) Add 2mL of urine (previously boiled and cooled to avoid
false
positive reaction).
(3) The appearance a green or blue color with in 5minutes
indicates
the presence of blood.
Report as :
Trace = faint green
1+ = green
2+ = greenish blue
3+ = blue
4+ = deep blue
09/28/22 127
Occultest (tablet method)
Principle
When the tablet is moistened with water tartaric acid
and calcium acetate react with strontium peroxide to
form hydrogen peroxide.
The hemoglobin in the urine catalytically decomposes
hydrogen peroxide liberating oxygen, which oxidizes
orthotolidine to a blue derivative.
09/28/22 128
Procedure
09/28/22 129
Reagent Strip Tests (Hemastix)
Principle:
The hemoglobin test spot of the reagent strip contains a
buffered mixture of organic peroxide and a chromogen
(o-toluidine, this is different from o-tolidine).
The color of the chromogen changes in presence of
hemoglobin.
Presence of high concentration of ascorbic acid or
vitamin C in a patient’s urine may inhiabit or delay the
color reaction for hemoglobin as the ascorbic acid acts as
the oxygen acceptor instead of the chromogen
09/28/22 130
Procedure
09/28/22 131
Interpretation
09/28/22 132
Clinical significance
09/28/22 133
Cont…
09/28/22 134
Urine Myoglobin
09/28/22 135
Myoglobin Cont’D…
09/28/22 136
Clinical Significance
09/28/22 137
09/28/22 138
Determination of Bilirubin
09/28/22 139
Production of Bilirubin
09/28/22 140
Production of Bilirubin
09/28/22 141
Production of Bilirubin
09/28/22 142
Production of Bilirubin
09/28/22 143
09/28/22 144
Clinical Significance
Conjugated bilirubin appears in the urine when the
normal degradation cycle is disrupted by obstruction of
the bile duct (e.g., gallstones or cancer) or when the
integrity of the liver is damaged, allowing leakage of
conjugated bilirubin into the circulation.
Hepatitis and cirrhosis are common examples of
conditions that produce liver damage, resulting in
bilirubinuria.
Jaundice due to increased destruction of red blood cells
does not produce bilirubinuria. This is because the
serum bilirubin is present in the unconjugated form and
the kidneys cannot excrete it.
09/28/22 145
Methods of determination of bilirubin
09/28/22 146
Harrison’s (Fouchet’s) Test
Principle
Barium chloride reacts with sulphate radical in the
urine to form barium sulphate.
If bilirubin is present in the urine, it adheres to the
precipitate and is detected by the oxidation of bilirubin
(yellow-colored) to biliverdin (green-colored) on
treatment with ferric chloride (Fouchet’s reagent) in the
presence of trichloracetic acid.
A blue color is given by bilicyanin.
09/28/22 147
Harrison’s (Fouchet’s) Test Procedure
Positive: Yellow
Check the pH of the urine with a litmus paper strip.
It should be slightly acidic.
If alkaline, acidify with a few drops of 33% acetic acid.
Take 10ml of the centrifuged urine and add to that 5ml
of 10% barium chloride.
Mix well.
It will become milky with white or yellow precipitate. If
the precipitate formed is insufficient, add a drop of dilute
sulphuric acid or ammonium sulphate solution.
Filter the precipitate through a small size filter paper .
09/28/22 148
Procedure Cont…
take the paper out and carefully unfold it on a dry filter
paper to soak the fluid and place both on a white tile or
wax paper.
Note the color of the precipitate and make the preliminary
observation:
Negative: White or nearly white
Add one drop of Fauchet’s reagent onto the precipitate in
the center of the filter paper holding the precipitate.
If bile is present, a green or blue color develops; the
color intensity is proportional to the amount of bile
pigment present.
Negative: No change in color
Pale blue-green:Trace or 1+
Darker blue-green: 2+ to 4+
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Barium chloride filter paper method
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Diazotization Test for bilirubin
Principle:
the tablet and reagent strip tests for bilirubin are
based on the coupling of bilirubin with a diazonium
salt (p-nitrobenzene diazonium p-toluen sulfonate) in
an acid medium to form azobilirubin, which gives a
blue or purple color.
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Reagent Strip (Diazo) Reactions
testing for urinary bilirubin by reagent strip uses the diazo reaction.
Bilirubin combines with 2,4-dichloroaniline diazonium salt or 2,6-
dichlorobenzene-diazonium-tetrafluoroborate in an acid medium to
produce an azodye, with colors ranging from increasing degrees of
tan or pink to violet, respectively.
Qualitative results are reported as negative, small, moderate, or
large, or as negative, 1, 2, or 3.
Reagent strip color reactions for bilirubin are more difficult to
interpret and are easily influenced by other pigments present in the
urine.
bilirubin glucuronide + diazonium salt → azodye
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Icto test Tablet test
Principle
Ictotest tablet test is based on a diazo reaction, in
which bilirubin is coupled with a diazonium salt in an
acid medium.
The tablets are supplied with a special absorbent mat.
The tablet contains the reactive ingredients.
The tablet is place over the urine on the mat, and
water is added to the tablet.
When bilirubin is present, it reacts with a solid
diazonium salt present in the tablet to form azobilirubin.
A positive reaction is seen as a blue or purple color on
the mat.
.
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Procedure
Place 10 drops of urine on the centerl test mat
Place the Ictotest tablet in the center of the urine-moistened area.
Place 1 drops of water onto the tablet. Wait 5 seconds; then place
a second drop of water onto the tablet so that the water runs off the
tablet onto the mat.
Remove the tablet, and observe the mat for the appearance of a
blue to purple color at 60 seconds.
Negative: The mat shows no blue or purple within 60
seconds.
Positive: The mat around or under the tablet turns blue or
purples, within seconds.
Sensitivity:Ictotest detects 0.1mg of bilirubin in 100ml of urine.
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Determination of Urobilinogen
Introduction
Inthe intestine, most of the bilirubin is converted to
urobilinogen or stercobilinogen by the action of certain
bacteria that make up the intestinal flora.
Approximately half of the urobilinogen formed in the
intestine is absorbed into the portal blood circulation
and returned to the liver.
In the liver most of the urobilinogen is excreted into
the bile once again and returned to the intestine.
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Urobilinogen int. cont’d…
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Urobilinogen int. cont…
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Urobilinogen int. cont…
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Test for urobilinogen
EhrlichTest
Wallace-Diamond quick screen method
Reagent Strip Tests for urobilinogen-uroblistix
Semiquantitation of urobilinogen
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Ehrlich Test
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Ehrlich Test cont’d…
Procedure:
1. Place 10 ml urine in a test tube.
2. Allow warming to room temperature.
3. Add 1 ml Ehrlich's reagent and mix.
4. Let stand 3 to 5 minutes
Normal amounts of urobilinogen present in the urine
sample will change the solution to pink.
Abnormally high amounts of urobilinogen will change
the solution to a Cherry red color.
This must be reported positive for urobilinogen
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Ehrlich Test (cont…)
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Clinical Significance
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Test For Urobilin
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Schilesinger's Test for Urobilin
Principle:
When zinc acetate reacts with urobilin it produces a
green fluorescence.
Reagent
Alcoholic solution of zinc acetate for urobilin.
Place 100ml of Ethyl in a beaker.
Add zinc acetate with strong until no more goes in to
solution.
Weigh 5gm of Iodine and 10gm of potassium Iodid.T
transfer all reagents to a brown bottle.
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Porphobilinogen
Introduction:
Porphobilinogen is a normal, colorless precursor of the
porphyrin.
Porphyrin are a group of compounds used in the
synthesis of hemoglobin.
Porphyrins are normally eliminated from the body in the
urine and feces primarily as coprophyrin I with a small
amount of coproporphyin III.
However, with certain inherited enzyme deficiencies
such as lack of porphobilinogen deaminase, there is
blockage in the normal pathway with increased
excretion of other porphyrins in the urine
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Hoesch Test for Porphobilinogen
Principle:
This is a test for porphobilinogen based on an inverse
Ehrlich’s aldehyde reaction.
In the Hoesch test, an acid solution is maintained by
adding a small volume of urine to a relatively large
volume of Ehrlich reagents.
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Hoesch Test for Porphobilinogen
cont’d…
Reagent
1. Hydrochloric acid, 6mol/L.
Dilute concentrated Hcl 1:2 with deionized or distilled
water by slowly adding the acid to water.
2. Hoesch reagent:
Dissolve 2.0g P-dimethylaminobenzaldehyde in 6
mol/L Hcl & dilute to 100ml with Hcl.
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Clinical Significance
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Leukocyte Esterase
Introduction:
Measurement of leukocyte esterase is an indirect
measurement of leukocytes.
Positive leukocyte esterase reactions in urine occur
most often with increased neutrophiles, which are
present in response to bacterial infections.
The lymphocytes and epithelial cells do not certain
leukocyte esterase and are not measured in this test.
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Leukocyte Esterase int. cont’d…
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Reagent strip test for leukocyte esterase
Principle
Reagent strip tests utilize a diazo reaction. The test
area contains an ester that is hydrolyzed by leukocyte
esterase to form its alcohol (which contains an
aromatic ring) and acid.
The aromatic ring is then coupled with a diazonium
salt, present in the test area, to form an azo dye, which
is seen as the formation of a purple color.
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Clinical Significance
When the urinary tract is infected at any point from the
urethra to the kidney, increased numbers of leukocytes,
especially neutrophils, are typical.
These cells are also seen in the urinary sediment.
However, neutrophils are rapidly lysed or destroyed in
the urine, possibly a result of their phagocytic activity.
Once lysed, they are undetectable in the microscopic
examination of the sediment.
However, the reagent strip tests for leukocyte esterase
remains positive whether lysed or intact cells are in the
urine.
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Clinical Significance
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Determination of Nitrate
Reagent strip tests for nitrite are used as a rapid method
for detecting asymptomatic UTI.
This test is most useful when combined with reagent
strip tests for leukocyte esterase.
The detection of nitrite in the urine can be used to
indicate the presence of bacteria that reduce urinary
nitrate to nitrite.
The presence of urinary nitrite indicates the existence of
a urinary tract infection.
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Method Used for Detection of Nitrite
Microscopic examination:
urine sediment when examined microscopically can
reveal bacteria when present.
Chemical dipstick
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Reagent Strip Test
Principle
Reagent strip test for nitrate are based on the Griess
test.
This involves a diazo reaction. Nitrate will read with an
aromatic amino (P- arsanilic or sulfanilic acid) in an acid
medium to produce a diazonium salt.
The diazonium salt is then coupled with another
aromatic ring (quinoline) to give an azo dye, which is
seen as a pink or red color.
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Reagent Strip Test cont’d
Limitation:
False positive reactions may be caused by bacterial
growth in ‘old’ urine specimen or by medication such
as Phenazopyridine that colors the urine red or that
turn red in an acidic medium.
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Interference
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Interference cont’d…
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Clinical Significance
Most infections begins in the lower urinary tract as a
result of fecal contamination from the organism
normally present in the feces, such as E.coli
The urinary tract is normally sterile.
Infection is introduced via the urethra and ascends to
the bladder, ureters, and finally the kidney.
Early detection and treatment is important to prevent
this ascending infection and subsequent renal
involvement.
Detection of such infection is particularly important in
pregnant women and young girls to prevent
permanent renal damage.
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Determination of Urinary Calcium
Introduction:
vitally important in maintaining the correct conditions for
normal neuromuscular transmission and glandular
secretion and for the activity of enzyme systems
particularly those involved in blood coagulation.
The bulk of calcium ions (Ca++) discharged by body is
excreted in the stool. However, there is small quantity
of calcium that is normally excreted urine.
But it may increase depending up on the quantity of
dietary calcium ingested.
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Calcium Test (Sulkowitch Test)
Principle
In this test a solution of ammonium oxalate ( about
4 % ) is added to the urine. If calcium is present in
excessive amounts, it drops out of solution as a heavy
white precipitate of calcium oxalate.
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Procedure
1.Pour 5 ml of urine into a test tube
2. Add 5 ml of Sulkowitch reagent
3. Mix by inverting the tube several times
4. Allow to stand 3 minutes
5. If a fine white cloud appears, the calcium content is
normal
6. If no white cloud appears, the calcium content is
decreased
7. If a heavy white milky precipitate forms, the calcium
content is increased
8. Report the calcium content as normal, decreased or
increased
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Interfering factors
False positive
High sodium and magnesium intake
High milk intake
If test done immediately after high calcium meal
False negatives
Increased dietary phosphates
Alkaline urine
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Clinical Significance
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Determination of Indican
Introduction:
Indican is derived from indole, which is the
putrefaction product of protein, by protolytic bacteria.
Patients with bacterial over growth in the small
intestine excrete large amount of metabolites of amino
acids such as tryptophan or tyrosine.
Indole is produced by bacterial action on tryptophan
in the intestine, which mostly eliminated with feces,
some are absorbed and detoxified in the liver and
excreted as indican in the urine.
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Indican cont…
It is increased in:
High protein diet
Pathological conditions such as
Bacterial putrefactions.
Enteritis.
Pancreatic insufficiency.
Intestinal Infection.
Ulceration of intestinal mucosa.
Note: To differentiate the pathological conditions from non-
pathological, first restrict the patient from protein intake and
then do the test.
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Indican Tests ( Obemyares Test)
Principle:
HCL liberates indoxyl from indican and ferric chloride
(FeCl3) oxidizes the indoxyl to indigo blue.
09/28/22 191
Procedure
09/28/22 192
Interpretation
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Source of Errors
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Determination of Melanin
Introduction:
Melanin is pigment derived from tyrosine, which is normally present
in hair, skin and in the choroid layer of the eye
Melanomas with pigments are normally transferred from
melanocytes to skin and mucus membrane cells.
In patients with tumors arising from the melanin producing cells, the
melanomas, the melanin may be excreted in the urine in large
amount, and its presence is indicative of metastasis of the tumor to
the liver or other organ.
The urine becomes black upon standing (oxidation), where the
chromogen /melanogen is changed into the pigment called melanin
which is a physical method to detect melanin.
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Chemical Tests for Melanin
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Clinical Significance
09/28/22 197
Chemical Tests for Melanin cont’d
A. Ferric Chloride Test
Principle:
FeCl3 oxidizes the melanogen to melanin.
Procedure
1.To 5 ml of freshly voided urine, add 1 ml 10% FeCl3 drop
by drop to precipitate all the phosphates.
2. Add drop by drop 10% HCl to dissolve all the precipitate
and it forms different color.
3. Centrifuge and examine for black or gray precipitate of
melanin.
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Chemical Tests for Melanin cont’d
B. Thormahlel Test
Principle
Sodium Nitroprusside is reduced to ferocyanide
(Prussian blue) by reducing action of melanogen.
Procedure
09/28/22 200
ASCORBIC ACID
Introduction:
Ascorbic acid (vitamin C) is neither a normal nor a
pathologic constituent of urine.
Ascorbic acid interfere with the various reagent strip
tests that are based on the diazo reaction.
In these tests, the ascorbic acid may react with the
diazonium salt that is formed, causing a reduced or
false-negative reaction.
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Reagent Strip Test for Ascorbic Acid
Principle:
The reaction is based on the reduction by ascorbic
acid of phosphomolybdate, a yellow complex, to
molybdenum blue.
Specificity
The reaction is not specific for ascorbic acid but will
react with other reducing substances with similar
redox potential
09/28/22 202
Mucopolysaccharide (MPS)
Introduction:
Mucopolysaccharidosis in infants may lead to dwarfism
and mental retardation.
In this genetic disorder mucopolysaccharide (a
conjugated protein with carbohydrate) is found in the
urine.
Special test-strip is available for testing the presence of
mucopolysaccharide in urine.
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Exercises
09/28/22 204
References:
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205
The next chapter will be on
Microscopic Examination Of Urine