GMELIN’S TEST

1. Outline the steps in the degration of

hemoglobin to bilirubin, urobilinogens,
and, finally, urobilin.
■ Urobilin is generated from the degradation of heme, which is
first degraded through biliverdin to bilirubin. Bilirubin is then
excreted as bile, which is further degraded by microbes
present in the large intestine to urobilinogen. Some of this
remains in the large intestine, and its conversion
to stercobilin gives feces its brown color. Some is
reabsorbed into the bloodstream and then delivered to
kidney. When urobilinogen is exposed to air, it is oxidized to
urobilin, giving urine its yellow color.
 2. State the reasons behind increased and
decreased urine urobilinogen levels.
■ Two situations can lead to an increase in urobilinogen levels in urine: a
liver disease that disturbs the normal passage of urobilinogen through
the liver and gallbladder (viral hepatitis, cirrhosis of the liver, obstruction
of the gallbladder by gallstones, etc.), or a urobilinogen overload caused
by the release of larger quantities of hemoglobin (destruction of
abnormally high number of red blood cells as in hemolytic anemia,
pernicious anemia, etc.).
■ Low urine urobilinogen levels may result from congenital enzymatic
jaundice (hyperbilirubinemia syndromes) or from treatment with drugs
that acidify urine, such as ammonium chloride or ascorbic acid.
Describe the relationship of urinary
bilirubin and urobilinogen with the
diagnosis of bile duct obstruction, liver
disease, and hemolytic disorders.
■ Urobilinogen is formed from the reduction of
bilirubin. Bilirubin is a yellowish substance found in
your liver that helps break down red blood cells.
Normal urine contains some urobilinogen. Too
much urobilinogen in urine can indicate a liver
disease such as hepatitis or cirrhosis.
4. Compare the principle involved in the
procedure performed with the reagent
strip test for urinary bilirubin, including
possible sources of error.
■ Samples suspected of containing bilirubin should be
handled cautiously because of the possibility of
hepatitis. These samples should also be protected
from light until testing is completed, since direct
light will cause decomposition of bilirubin. The test
for bilirubin is based on the coupling of bilirubin with
a dye to form a color.
BLOOD: GUAIAC’S
TEST
Step 1

■ Label 2 clean and dry test tube.

Step 2

■ Mix 1.0 mL guaiac solution and 1.0 mL of “old

oil turpentine” together.
Step 3

■ Layer on this solution about 0.5 mL of

urine samples.
Specific Objective: A test for presence of
blood in urine and other samples such as
stool.
 Observation Normal Urine Sample Abnormal Urine Sample

Color of Solution Honey-like/Light Brown Color Honey-like/Light Brown Color

Interpretation (Positive or Negative) Negative Negative

1. How do hematuria, hemoglobinuria,
and myoglobinuria differ with regard to
the appearance of urine and serum and
its clinical significance?
 Description Appearance Clinical Significance

presence of blood in the urine in the form of intact

red blood cell

Hematuria Cloudy Red Urine Renal or Genitourinary

presence of blood in the urine as the product

of red blood cell destruction

Lysis of red blood cells produced

Hemoglobinuria Clear Red Specimen
in the urinary tract

Heme-containing protein found in muscle tissue it

results to muscle destruction (rhabdomyolysis)

Clear Reddish Brown Cholesterol lowering satin

Myoglobinuria
Urine medications
2. Compare the principle involved in the
Guaiac’s test with the reagent strip method
for blood testing, and list possible causes of
interference.
REAGENT STRIP REACTIONS

■ In the presence of free hemoglobin/myoglobin,

uniform color ranging from a negative yellow
through green to a strongly positive green-blue
will appear on the pad. The degree of
hematuria can then be estimated by the
intensity of the speckled pattern.
REACTION INTERFERENCE:

■ FALSE-POSITIVE
- menstrual contamination
- Strong oxidizing deter- gents are present in the specimen
container.
- Vegetable peroxidase and bacterial enzymes, including an
Escherichia coliperoxidase,
- Ascorbic acid
GUAIAC TEST

■ A test for blood in urine or feces using a

reagent containing guaiacum that yields a blue
color when blood is present. Some food scan
affect test results.
REACTION INTERFERENCE:
Do not eat the following foods for 3 days before the test:
■ Red meat
■ Cantaloupe
■ Uncooked broccoli
■ Turnip
■ Radish
■ Horseradish
■ Some medicines may interfere with the test. These include vitamin C, aspirin, and
non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen and
naproxen. Ask your doctor or nurse if you need to stop these before the test.
3. Discuss the methods used to
differentiate between hemoglobinuria
and myoglobinuria.
 The diagnosis of myoglobinuria is usually based on the patient’s
history and elevated serum levels of the enzymes creatinine kinase and
lacticdehydrogenase. The appearance of the patient’s plasma also can
aid in the differentiation. The kidneys rapidly clear myoglobin from the
plasma, leaving normal appearing plasma, whereas hemoglobin bound to
haptoglobin remains in the plasma and imparts a red color. The principle
of this screening test is based on the fact that the larger hemoglobin
molecules will be precipitated by the ammonium sulfate, and myoglobin
will remain in the supernatant. Therefore, when myoglobin is present, the
supernatant will retain the red color and give a positive reagent strip test
for blood. Conversely, hemoglobin will produce a red precipitate and a
supernatant that tests negative for blood. Myoglobin is not stable in acid
urine and, if denatured, may precipitate with the ammonium sulphate.
Never stop or change your medicine without first talking to your
healthcare provider.
ELECTROLYTES
1. Flame test: Sodium Ion
Step 1
■Dip a cleaned test wire (clean in 3M
HCl) into each urine specimen.
Step 2

■ Place the wire loop in the flame and

record the color of the flame
Specific Objective: To determine the
presence of sodium ion.

Normal Urine Abnormal Urine

Observation
Sample Sample

Color of Orange Orange

Flame (Positive) (Positive)
1. What is a flame test? How are flame
colors produced?
■ Flame tests are utilised in chemistry to identify the metal ions in
compounds. They are more useful for some metals than others;
particularly for the Group 1 metals, they provide a good way of quickly
identifying the metal ion present.
To carry out the flame tests, a small amount of the compound
being tested will be held in a flame and the colour given off observed.
This colour originates from the movement of electrons in the metal ion.
When heated, the electrons gain energy, and are ‘excited’ into higher
energy levels; however, the electrons occupying these levels is more
energetically unstable, and they tend to fall back down to their original
energy levels, releasing energy as they do so. This energy is released as
light, with the characteristic flame colours of different metal ions due to
varying electron transitions.
2. Discuss the various flame colors of
different ions.
What is the purpose of dipping the copper
wire into the acid before subjecting it to
flame?
■ Dipping the copper wire into the acid (HCl)
is a way of cleaning the copper wire from any
elements that would affect the change of color
in the flame test.
Silver Nitrate Test: Chloride ion
Step 1
■Place 3mL of each urine specimen in
a separate tube.
Step 2

■ Add 5 drops of 3M HNO3 and 5 drops of

0.1 M AgNO3. Mix thoroughly.
Specific Objective: To determine the
presence of chloride ion.

Normal Urine Abnormal Urine

Observation
Sample Sample

Color of
White (Positive) White (Positive)
Precipitate
1. What is the role of nitric acid in the test
for chloride?
■ Silver nitrate precipitates many anions.
Nitric acid is added to prevent the
precipitation of anions other than
chlorides.
2. Why should the specimen be albumin-
free when performing the chloride test?

■ Albumin is a type of protein. And protein is

usually broken down into urea before
going to mix with urine. If albumin is not
broken down, the test may also give a
false positive result because albumin may
interfere with the reaction.
3. Write the chemical equation involved in
the test.
DIPSTICK TESTS
Normal Urine Diabetic Urine
1. Why is it important to time your
observation of test strips carefully after
dipping them in the sample tested?
■ To have a appropriate results and so that the
reagents might have an ample time to react
with the urine.
2. List 4 causes of premature deterioration
of reagent strips, and explain how they can
be avoided.
■ Moisture
■ Volatile Chemicals
■ Heat
■ Light
3. List 5 quality-control procedures routinely
performed with reagent strip testing.
■ 1. Test open bottles of reagent strips with known
positive and negative controls every 24 hr.
■ 2. Resolve control results that are out of range by
further testing.
■ 3. Test reagents used in backup tests with positive
and negative controls.
■ 4. Perform positive and negative controls on new
reagents and newly opened bottles of reagent strips.
■ 5. Record all control results and reagent lot
numbers.
4. Discuss the principles of the reagent
strip method for the different reaction
(chemical test) and incorporate your answer
with the tests above.
Urine Home Urobilinogen Test
Norm. Norm. 2 4 8
0.2 1

Wait 60 Seconds - Expected result: less than 17

µmol/l (< 1mg/dl) - mg/dL Urine (1 mg = approx. 1
EU) - Urobilinogen: Urine Urobilinogen Test: A
urobilinogen in urine test measures the amount of
urobilinogen in a pee sample. The test should be
carried out at room temperature as the reaction's
sensitivity increases with temperature.
Home Urine Glucose Test
Neg. 1/10 (ir.) 1/4 1/2 1 2+
100 250 500 1,000 2,000+

Wait 30 Seconds - g/dL (%) - Expected result: Negative - In order to

measure the amount of sugar in your urine, you need a urine test strip
(dipstick) and a container for collecting urine. It is important to talk
with your doctor about the best time of day to perform the urine
glucose test, and whether to do it before or after eating.
Home Urine Bilirubin Test
Neg. Small Mod. Large
+ ++ +++

Wait 30 Seconds - Expected result: Negative - Bilirubin is a

highly pigmented compound that is a by-product of
haemoglobin degradation.
Home Urine Ketone Test
Neg. Trace Small Mod. Large Large
5 15 40 80 160

Wait 40 Seconds - Expected result: Negative - Ketone: This

urine test measures ketone levels in your pee.
Home Urine Specific Gravity Test
1.0 1.005 1.010 1.015 1.020 1.025 1.030

Wait 45 Seconds - Expected Results: low at 1.000 but normal ranges from
1.005- Specific Gravity
Blood in Urine Home Test
Neg. Few Many Trace Small Mod.
Dark Flecks Dark Flecks + ++

Wait - 60 Seconds - Expected result: Negative - Blood in the urine is called

hematuria. Blood present in urine in large quantities can be detected
visually.
Home Urine pH Level Test
5 6 6.5 7 7.5 8

Wait 60 Seconds - Expected result: Low - The pH level indicates the

amount of acid in urine.

Home Urine Protein Test

Neg. Trace

Wait 60 Seconds - Expected result: Negative - Protein mg/dL : This test is

usually run to screen for kidney disease.
Home Urine Nitrites Test
Neg. Pos. Pos.
Any Pink Any Pink

Wait 60 Seconds - Expected result: Negative - Nitrite

Urine Leukocytes Home Test
Neg. Trace Small Mod. Large

Wait 2 Minutes - Expected result: Negative - Leukocytes

Principles of Chemical Tests
pH - The pH is a measure of the degree of acidity or alkalinity
of the urine. A pH below 7 indicates an acid urine; pH above 7
indicates an alkaline urine. Normal, freshly-voided urine may
have a pH range of 5.5 - 8.0. The pH of urine may change with
diet, medications, kidney disease, and metabolic diseases
such as diabetes mellitus. Colors on the pH reagent pad
usually range from yellow-orange for acid pH to green-blue
when pH is alkaline.
Protein - Protein in the urine is called proteinuria. This is an
important indicator of renal disease, but can be caused by
other conditions as well. At a constant pH, the development of
any green color on the protein reagent pad is due to the
presence of protein. Colors range from yellow for negative to
yellow-green or green for positive.
Glucose - The presence of glucose in urine is called glycosuria.
This condition indicates that the blood glucose level has
exceeded the renal threshold. This condition may occur in
diabetes mellitus. The reagent strip is specific for glucose and
uses the enzymes glucose oxidase and peroxidase, which
react with glucose to form colors ranging from green (low
concentration) to brown (high concentration.
Ketone – Ketones present in the blood is known as ketonuria.
This occurs when the body metabolizes fats incompletely
causing ketones to be excreted in the urine. The ketone test is
based on the development of colors ranging from light pink to
maroon when ketones react with nitroprusside. Ketonuria may
be present in diabetes, starvation or fasting. Since ketones will
evaporate at room temperature, urine should be tightly
covered and refrigerated if not tested promptly.
Bilirubin – Bilirubin in the urine is known as bilirubinuria.
Bilirubin is a breakdown product of hemoglobin which
produces an extremely yellow to amber color in urine and may
be an indication of liver disease, hepatitis or bile duct
obstruction. Samples suspected of containing bilirubin should
be handled cautiously because of the possibility of hepatitis.
These samples should also be protected from light until testing
is completed, since direct light will cause decomposition of
bilirubin. The test for bilirubin is based on the coupling of
bilirubin with a dye to form a color.
Blood - Hemoglobin and red blood cells in urine may be
detected by the formation of a color due to the enzyme
peroxidase (in red cells) reacting with orthotoluidine, a
chemical which is in the reagent pad. The resulting color
ranges from orange through green to dark blue.
Hemoglobinuria is the presence of hemoglobin in the
urine. Causes: hemolytic anemia, blood transfusion
reactions, massive bums, renal disease
Leukocytes - Leukocytes (aka white blood cells) present
in large numbers usually indicate a urinary tract
infection (UTI). Normal urines should produce no color
change of the Leukocyte pad.
Nitrites - This test indicates the conversion of nitrate to
nitrite by the action of certain bacteria in the urine. A
positive result indicates a possible UTI and the
potential need for a culture.
Specific Gravity - The specific gravity of a solution is the ratio
of the weight of a given volume of the solution (urine) to the
weight of an equal volume of water. The specific gravity of
urine indicates the concentration of dissolved solids such as
urea, phosphates, chlorides, or proteins present in the urine.
Normal specific gravity is 1.005 - 1.030 with most normal
results falling between 1.010 and 1.025. The higher the
number the more concentrated the urine. Test for water-loss
dehydration.
Ascorbic Acid – Concentrations of ascorbic acid as low as 20
mg/dL interfere with enzyme-driven reactions on the dipstick.
The presence of ascorbic acid will result in falsely decreased
readings of glucose, nitrite and blood. (Note – not all dipstick
manufacturers offer this test on their product.)
Substance Reference Ranges (aka Normal Values)

pH 5.5 - 8.0

Protein negative to trace

Glucose Negative

Ketones Negative

Bilirubin Negative

Blood Negative

Urobilinogen 0.1 - 1.0 EU/dl – do NOT report as negative!

Leukocytes Negative

Nitrites Negative

Specific Gravity 1.005-1.030

 5. Why is the reagent strip method more
accurate and sensitive than other chemical
tests?
■ It is more accurate than other chemical tests
because when you do the process in reagent strip
method correctly you will instantly see the result
using the parameters in the reagent strip bottle and
it is more sensitive from other chemical tests
because one mistake will make the process
erroneous.