ANALYSIS OF URINE AND BODY FLUIDS LAB

Lecturer: FERLY PEÑAVERDE, ALYSSA LAGON AUGUST 2022

ANALYSIS OF URINE AND BODY FLUIDS LABORATORY

MODULE 1: MICROSCOPY
OUTLINE

I. Microscopy V. Determination of MICROSCOPY

A. Types of Proteins in Urine
Microscope A. Qualitative ● Microscope
B. Parts of B. Quantitative ○ an optical instrument that magnifies the size
Microscope VI. Test for Glucose of the image of the object.
C. Handling and (Dextrose) in Urine ○ used to observe objects that are not visible
Care A. with the unaided eye.
D. Manipulation VII. Detection of ○ It is probably the equipment that is the most
E. Magnification Ketone Bodies in used (and misused) in the clinical laboratory.
II. Collection and urine ○ It is indispensable in many areas of the
Preservation of VIII. Detection of Bile laboratory, including urinalysis, hematology,
Urine Specimen Pigments and microbiology.
A. Methods of Urine ○ The microscope is a precision instrument
Collection and is very important equipment thus, it must
B. Types of Urine be kept clean and in excellent condition,
Specimen mechanically and optically, at all times.
C. Methods of
Preservation of The invention of this magnifying instrument was
Urine credited to Galileo Galilei & Zacharias Janssen and
III. Physical its use started with Anton Van Leuwenhoek.
Characteristics of
Urine Two Types of Microscope
a. Color
b. Odor 1. Simple microscope- has a single biconvex
c. Specific Gravity magnifying lens
d. Volume 2. Compound microscope- has 2 or more lenses
IV. Screening Tests a. Light microscope- employs visible light as
For Chemical source of illumination
Components Of b. Electron microscope- source of illumination
Urine (Reagent is beams of electrons
Strip Method
a. Protein Parts of the Microscope
b.Glucose Classified into four systems (based on function)
c.Blood
d.Ketone Adjustment System
e.Bilirubin
1. Coarse adjustment knob- moves the stage up and
f.Urobilinogen
down (scanner and LPO)
g.Nitrite
2. Fine adjustment knob- brings the image into sharp
h.Leukocytes
focus ( HPO and OIO)
i.pH
Support System
Base- supports the microscope
Arm- supports the body/tube and connects it to the
base
Revolving Nosepiece- holds the objectives and can
be rotated to easily change power
Stage- horizontal platform with central opening

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
Mechanical Stage- gives controlled movement to the
object on the slide
Magnification System
1. Objectives- one or more lends held in position by a
hollow metal cylinder; magnifies the object.
a. Scanning -4x (red)
b. Low power- 10x (yellow)
c. High power (retractable)- 40x (blue)
d. Oil immersion (retractable)- 100x (white)
2. Eyepiece - consists of upper eye lens and lower field
eye lens (usually 10x or 15x)
Illumination System
Source of light (daylight or electric light)
1. Mirror- reflects light rays from light source to the
object.
a. Concave mirror- used for near light source
b. Plane mirror- used for distant source of
lighted daylight
2. Condenser- focus the light to the specimen
3. Diaphragm- regulate the amount of light passing into
the condenser
4. Filter- contain blue and white filter below the
condenser
Handling and Care
1. Carry the microscope in an upright position with two
hands, one hand supporting the base & the other
hand grasping the arm.
2. Place the microscope carefully on the working table,
about 1 inch away from the edge of the table.
3. Remove the dust from the microscope using a soft
brush (preferably camel’s hair) or you may blow it
away. Only after this should the lenses be cleaned
with lens paper
4. Clean the eyepiece lens with dry lens paper and the
objective lenses with lens paper or cotton (dry or
moistened with xylene or 95% ethyl alcohol)
5. If the microscope is equipped with an electric cord,
check if it is in good condition before plugging into the
outlet.
6. Make sure that the scanner or the low power objective
(LPO) is in the focusing position. Always start each
new observation at the scanner or LPO.
7. If your microscope is equipped with a mirror, use a
lamp or sunlight as a source of light.
Note: Sunlight should not be allowed to fall directly on
the mirror to avoid damage to the user’s eyes, lenses
or slide. The best light is that which is reflected from
the clouds
Manipulation
1. Place the slide carefully on the stage and secure it
with stage clips or brackets.
2. Move the slide so that the specimen is at the center of
the stage hole.
3. While looking through the ocular, adjust the position of
the mirror so that it reflects light onto the specimen.
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4. Adjust (close/open) the diaphragm and (raise or
lower) the condenser until the microscopic field (the
circle of light seen through the eyepiece) is evenly
illuminated.
5. Looking at the stage, from the side, use the coarse
adjustment knob to raise the stage with the slide
toward the objective.
6. Then look through the ocular, with both eyes open,
7. Slowly rotate the coarse adjustment knob to move the
stage with the slide away from the objective.
8. When the image becomes visible, rotate the fine
adjustment knob to make the image appear sharper
and clearer.
Note: Never move the stage and the objective towards each
other while looking through the ocular and while focusing under
the high power objective (HPO) to avoid damaging the slide &
the objective lens.
9. Adjust the light intensity, using the lamp controller or
the diaphragm until the details of the object are at its
clearest.
10. Switch to HPO by rotating the revolving nosepiece.
Note: Most microscopes are parfocal, (the image
remains in focus even after shifting from one objective
to another) however, minor adjustment using fine
focus/adjustment knob is usually needed.
11. Switch to oil immersion objective (OIO) by rotating the
nosepiece halfway, then put a drop of oil on the slide
and rotate slowly until the OIO touches the oil drop
and it is in position. Slight rotation of the fine
adjustment/focus knob may be needed to clearly see
the details of the image/object in focus.
12. Record your observation by making a detailed
drawing of the prepared slide specimen.
13. After the observation, before removing the slide,
make sure that the LPO is in viewing position, the
objective and slide are as far apart as possible.
-switch off the light then turn the lever to 1 or 0 then
unplug the socket
14. Clean the objective lens with dry lens paper to
remove the oil followed by lens paper or cotton
moistened with a cleaning solution (xylene or 95%
ethyl alcohol).
15. Dispose the used cotton balls or lens paper in the
proper waste bin (labeled biodegradable).
16. Unplug the cord or adjust the mirror in a vertical
position and return the microscope to the shelf.
Note: Repeat the same procedure if you’ll examine other
slides
● Stage down when returning the microscope so move
the coarse adjustment
● If necessary, switch the microscope to storage
position
● Clean the slides with disinfectant and dry with lens
paper or just dry it with the lens paper
● Move the revolving nosepiece to scanner when
returning the microscope
2
 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY

Magnification Pathway of Light

1. Examine the magnification power of the ocular and

objective lenses. Write them in the table

Parts of the Brightfield Microscope

● Dust cover
○ helps protect the microscope and ocular
lenses
● Electrical cord
○ found at the back of your microscope
● Eyepiece
○ The lens the viewer looks through to see the
specimen. The eyepiece usually contains a
10X or 15X power lens.

● Diopter adjustment

○ Useful as a means to change focus on one

eyepiece so as to correct for any difference
in vision between your two eyes.
● Revolving nosepiece
● Objectives
● On/Off Switch
● Coarse adjustment knob
● Fine adjustment knob
● Stage Ɣ Stage clip
● Mechanical stage knob Figure 1. Pathway of Light
● Condenser
● Iris diaphragm Parts of the Microscope
● Condenser centering screws
● Field diaphragm- increasing/decreasing the light from
light source
● Light source

Notes:
● Do not touch the glass part of the lenses with your
fingers. Use only special lens paper to clean the
lenses.
● Always keep your microscope covered when not in
use.
● Always carry a microscope with both hands. Grasp
the arm with one hand and place the other hand
under the base for support
● Total magnification= Objective magnification x Ocular
magnification.
● The higher the magnification, the smaller the depth of
field
● Clean the stage with soft cloth and disinfectant (10%
hypochlorite) when there’s a spillage like urine and
semen
● DO NOT USE 10% hypochlorite solution in cleaning
the lenses
● Look into the ocular with both eyes to avoid eye strain Figure 1.2 Parts of Microscope
●

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
- Receiving sterile plastic bags are attached at
the side of the bed of the patient

MODULE 2: COLLECTION AND PRESERVATION OF

URINE SPECIMEN

METHODS OF URINE COLLECTION

● Gauze-pad Method - use for infants

- Infant urine bags - it has a stick on sticker
where the genital of baby is attached
- Clean the area surrounding the
genital of the baby using sterile
gauze
- Remove sticker and attach directly
at genital area
- Then, it will catch the urine
- 5 mL - maximum voided urine
- 60 mL - ideal amount of urine

● Suprapubic Aspiration (SPA) - urine may be

collected by external introduction of needle through
the abdomen into the bladder
- Sterile technique
- Also direct to urinary bladder
- Comatose patients
- Aspirate using plunger to void urine

● Catheterization- urethral catheter or ureteral catheter

- Sterile technique
- Plastic tubing directly inserted to the bladder
- Lubricated before inserted

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
● Mid- stream catch method/ Clean catch method
(no pic)
- Most common and widely used to detect
infection, do cell counts and culture &
sensitivity
- Medical Technologists must know how to
instruct patients on how to properly void
urine
- Instruct the patient to clear the genital area
- Void the urine and tell them to stop midway
- The first urine is dirty and mixed
with other cells
- Tell them to continue and catch the urine
4. Formalin (formaldehyde)- Acts as reducing agent
interfering with chemical tests for glucose, blood,
leukocyte esterase, & copper reduction
5. Toluene- Does not interfere with routine tests
6. Sodium Fluoride- Prevents glycolysis. Good
preservative for drug analyses
7. Commercial preservative tablets- Convenient when
refrigeration is not possible. Have controlled
concentration to minimize interference
8. Chloroform – inhibits bacterial growth, Aldosterone.
Causes change in the characteristics of the cellular
sediments.
9. Chlorhexidine – prevents bacterial growth & useful
as a glucose preservative.
10. 6N hydrochloric acid – for Vanillyl Mandelic
Acid/VMA
11. Sulfuric acid– for Cathecholamines
12. Usage of Gray C&S Tube – Sample stable at RT for
48 hrs. Preserves bacteria. Decreases pH.
13. Usage of Yellow Plain Tube – Use on Automated
instruments. Refrigerate w/in 2 hours.
14. Usage of Cherry Red Tube – Stable for 72 hours at
RT. Preservative is sodium propionate.
MODULE 3: PHYSICAL CHARACTERISTIC
S OF URINE

TYPES OF URINE SPECIMEN

COLOR
1. RANDOM – collected at any time ● Normal – varies from almost colorless, straw or light
2. CYTOLOGY STUDIES - with prior hydration yellow to dark yellow, yellow-orange, or amber
- For cell counts
3. FIRST MORNING/FIRST AVAILABLE SPECIMEN ● Variations -Colorless/pale yellow – recent fluid
(FAS) – First urine voided after sleep (6 to 8 hours). consumption, polyuria, DM, DI
Most concentrated urine.
- Not mid-stream/clean catch Amber/ orange bilirubin, acriflavine,
4. TIMED – Collect all urine during a specific timed phenazopyridine,nitrofura
interval. Examples are; 24 hours, 12 hours, 2 hours. ntoin, phenindione
- Urine collected for number of hours in one
container only
Yellow-green oxidation of bilirubin to
biliverdin
METHODS OF PRESERVATION OF URINE
There are two methods to preserve urine. One is through Blue/green Pseudomonas infection,
refrigeration which is a physical means to prevent bacterial amitriptyline,
growth and another one through addition of chemical methocarbamol, clorets,
preservatives. indican, methylene blue

The following are methods of urine preservation: Pink/red intact RBCs, hemoglobin,
myoglobin, porphyrins,
A. Refrigeration- most commonly used and precipitates beets, menstrual
amorphous phosphates & urates contamination
B. Addition of Chemical Preservatives - preserve
urine for further testing Brown/black methemoglobin,
homogentisic acid, melanin,
The following are examples of chemical preservatives used: argyrol methyldopa,
1. Thymol- Preserves glucose & sediments well levodopa,metronidazole
2. Boric Acid- Preserves protein & formed elements
well. Does not interfere with routine analyses other
than pH
3. Phenol- Does not interfere with routine tests ODOR

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
● Normal – faint aromatic due to volatile acids; ● Urinometer/Hydrometer - weighted float that is
becomes ammoniacal as the specimen stands designed to sink to a level of 1.000in distilled water;
calibrated at 20°C; less accurate than other methods;
requires large volume of urine
● Corrections
Variations: ■ Temperature – for every 3° that the urine
temperature is above or below the calibration
Ammoniacal (freshly UTI temperature, 0.001 is respectively add
voided) ■ ed to or subtracted from the reading
■ Protein – subtract 0.003 for every g/dL;
Rancid Tyrosinuria Glucose – subtract 0.004 for every g/dL B.
Refractometry 1) Refractometer/ TS meter -
Maple syrup/ caramel-like MSUD measures refractive index; compensated
between 15°Cand 38°CNote:- Has a float
Sulfur odor Cystine Cystine disorders that is designed to sink at 1.000 meter in
disorders -Fruity/ sweet distilled water.
- 1.000 - distilled water
Fruity/ sweet Diabetes ketoacidosis - 1.001+ - urine
- Requires a lot of urine sample
Mercaptan asparagus, garlic, and eggs - Should not stick to the side of the urinometer
- Read lower meniscus
Mousy PKU - Calibrated at specific temperature (disadvantage) -
check temp of urine before doing this method
Sweaty feet Isovaleric acidemia - Do correction for protein and sugar

Rotting fish Trimethyl

B. Refractometry
Fecaloid Recto-vesicular fistula ● Refractometer/ TS meter - measures refractive index;
compensated between 15°Cand 38°C
● Corrections: protein and glucose only; temperature
Cabbage/ hops Methionine malabsorption
correction not done
Bleach contamination
NOTE:The refractometer is calibrated using 1. distilled water
that should read 1.000. 2. 5% NaCl, should read- 1.022 ±
● TRANSPARENCY 0.001 3. 9% sucrose that should read 1.034 ± 0.001.

Normal- Clear – no visible particulates, transparent

Variations:

Hazy few particulates, print easily

seen through urine
C. Harmonic Oscillation Densitometry
Cloudy many particulates, print
blurred through urine ● Mass gravity meter – used by Yellow IRIS
automated workstations to measure specific gravity
Turbid print cannot be seen through ● Principle: Sound waves of specific frequency are
urine generated at one end of the tube and as the sound
waves oscillate through urine, their frequency is
Milky may precipitate or be clotted altered by the density of the specimen

VOLUME
SPECIFIC GRAVITY
Average daily output: 1,200 - 1,500 mL per day (1.2 L - 1.5 L)
● Normal Values: depend on the patient’s degree of
hydration Variations

METHODS Clinical significance:

A. Urinometry
Polyuria- abnormal increase Diabetes mellitus, Diabetes
in urine output insipidus

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY

Oliguria - abnormal Dehydration, renal

decrease in urine output insufficiency, poorly
compensated heart disease,
calculi formation, kidney
tumors

Anuria/Anuresis – total severe acute nephritis, Hg

suppression of urine Poisoning, obstructive
production uropathy, kidney failure

Nocturia – excretion of DM, DI

more than 500 mL urine at
night

Diuresis transitory increase in urine

volume 6. Milky

ADDITIONAL NOTES FROM VIRTUAL DISCUSSION

Specific Gravity - measure of amount of solute present in

urine
- High SG = high proteins, high precipitate
- Low SG = clear urine (except: diabetes insipidus)

7. Slightly Hazy
CLASS ACTIVITY: Clarity of Urine 8. Hazy
9. Cloudy
NOTE: 10. Turbid
WALA TA KABALO IF CORRECT TANAN TA NGA
ANSWER, GINA MINUSAN NYA LANG AGAD AGAD ANG
“IBAN” WITHOUT CORRECTION

INTERPRET THE DIFFERENT TRANSPARENCIES OF THE

FOLLOWING URINE SPECIMENS

11. Turbid
12. Hazy
13. Turbid

1. Hazy
2. Slightly Hazy

14. Turbid
15. Turbid

3. Slightly Hazy
4. Hazy
5. Turbid

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY

Tubes with Graduation

NOTE: Beaker
Sa Google: 7.) Clear 8.) Slightly Cloudy 9.) Cloudy 10.) Turbid

How to Measure Urine Volume

Visual - to measure
Specimen Cup

Test Tube

Graduated Cylinder

Pipettes (Serological)

Pasteur Pipette

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY

Specimen Cup
Volumetric Flask

Erlenmeyer Flask Pipettor

Burette

CLASS ACTIVITY: Volume of Urine

Reagent Bottle
(with graduation)

A. 37 mL
B. 37 mL
Syringe C. 40 mL
D. 65 mL
E. 2.3 mL

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY

Color Pathologic Non-pathologic

1.Green pseudomonas asparagus intake

infection
1. 440 mL
2. 7 mL 2.Orange dehydration too much carrots
3. 250 mL
4. 37 mL 3.Blue amitriptyline asparagus intake
5. 20 mL
4.Yellow Phenazopyridine
(Pyridium), urochrome

5.Yellow-green Bilirubin oxidized to excess vitamin

biliverdin B, B12

6. Red hematuria/presence beets

of RBC,

7. White excess fluid

(colorless) consumption,
6. 190 mL overhydration,
7. 86 mL diabetes insipidus
8. 35 mL
9. 280 mL 8. Black alkaptonuria,
10. 19 mL homogentisic acid,
tyrasonuria, large
amount of melanin
CLASS ACTIVITY: Reaction of Urine
1. pH 5.6 - Acidic
2. pH 8.3 - Alkaline
3. pH 7.1 - Slightly Alkaline
4. pH 6.3 - Acidic
5. pH 7.3 - Slightly Alkaline
6. pH 9.0 - Alkaline
7. pH 4.5 - Acidic
8. pH 6.8 - Acidic
9. pH 3.9 - Acidic
10. pH 8.0 - Alkaline

CLASS ACTIVITY: Colors of Urine

IDENTIFY THE COLORS OF THE URINE. NAME THE

CONDITIONS OR DISEASES ASSOCIATED WITH THE
COLORS

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY

Color Pathologic Non-Pathologi 20. Yellow Phenazopyridine

c (Pyridium),
urochrome
9.Portwine Porphyrins, liver
disease presence of
bile
Odor of Urine

10.Pale yellow normal color due to

urochrome

11.Pale yellow normal color due to

urochrome

12.Green pseudomonas asparagus

infection intake

13.Orange-Brow intake of
n anti-inflammatory
(amber) drugs (azulfidine),
hepatitis (presence of
bilirubin)

14.Red hematuria/presence Beets

of RBC,

15.Brown Homogentisic acid

(alkaptonuria)

16.Yellow-Green jaundice, liver excess vitamin

disorders B, B12

CLASS ACTIVITY: Odor of Urine

IDENTIFY THE CAUSES OF THE FOLLOWING ODORS OF

URINE

Odors of Urine Cause

Diabetes Mellitus

Color Pathologic Non-Pathologic

FRUITY/SWEET
17.Colorless overly hydrated

18.Brown/Tea hepatitis, liver

Colored (amber) disease, presence
of bile

Phenylketonuria
19. Dark yellow Dehydration

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY

MODULE 4: SCREENING TESTS FOR CHEMICAL

COMPONENTS OF URINE (REAGENT STRIP METHOD)

Reagent strips provide a simple, rapid means of

performing chemical analysis of urine including pH, Specific
gravity, Glucose, Protein, Ketones, Blood, Bilirubin,
Urobilinogen, Nitrite, & Leukocyte esterase. It consists of
MOUSY absorbent pads, impregnated with specific reagents, attached
to a plastic strip.

Note:
Specific reagents - react with the urine as you dip it
Methionine malabsorption
Chemical reaction takes place producing color (when
the absorbent pads come in contact with urine). These
reactions are interpreted by comparing the color produced on
CABBAGE-LIKE the pad with the chart supplied by the manufacturer.

Improper techniques can result in errors. Inaccurate

Isovaleric acidemia
SWEATY FEET
Bacterial Decomposition,
Urinary Tract Infection
interpretations may occur if the color reactions are read too
early or too late. For best semi-quantitative results, the
manufacturer's stated time should be followed. The use of
automated instruments in reading the results is recommended.
Strips must be protected from deterioration caused by
moisture, volatile chemicals, heat and light and care must be
taken not to touch the chemical pads when removing the strips
from the container. A good light source is essential for accurate
interpretation of color reactions. The strips must be held close
to the color charts without touching the chart. Reagent strips
and color charts from different manufacturers should not be
interchanged. Interferingsubstances in the urine, technical
carelessness, & color blindness also produce errors.

FOUL/AMMONICAL

CLASS ACTIVITY: Specific Gravity of Urine

INCREASED DECREASED

● A child with high ● After a night of

temperature alcohol party
PRINCIPLES
● A diabetic person ● Drinking tea
● From an ● A boy with low
edematous lady temperature PROTEIN (60 SECONDS)
● After strenuous ● Drinking 1.5 liter of The reaction is based on the phenomenon known as
exercise water the “protein error” of pH indicators, which means that the point
● A severely burned ● Drinking coconut color change of some pH indicators is different in the presence
patient water
of protein from that observed in the absence of protein,
because proteins act as hydrogen ion acceptors at a constant
pH. The development of any green color in the protein reagent
pad is due to the presence of protein at a constant pH of 3.

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
Colors range from yellow to yellow-green for negative contain enzymes known as esterases. Increased numbers of
results and green to green-blue for positive results. neutrophils usually indicate the presence of urinary tract
infection and their presence is indicated by a positive leukocyte
GLUCOSE (30 SECONDS) esterase test.
Reagent strip test reveals the presence of granulocyte
This test is based on enzymatic reactions between
esterases. The esterases cleave a derivatized pyrazole amine
glucose oxidase, peroxidase chromogen. Glucose oxidase
acid ester to liberate a derivatized hydroxy pyrazole.| This
oxidizes glucose to gluconic acid and at the same time reduces
pyrazole reacts with diazonium salt to produce beige pink to
atmospheric oxygen hydrogen peroxide. The hydrogen
purple color.
peroxide reacts with potassium iodide chromogen in the
presence of hydrogen peroxidase. The extent to which the
pH (60 SECONDS)
chromogen is oxidized determines the color which is
produced, ranging from green to brown. Reagent strips use a double indicator system of
methyl red and bromothymol blue that measure urine pH
BLOOD (60 SECONDS) between 5 to 9. The methyl red produces a color change
from red-orange to yellow in the pH range 4.4 to 6.2.
Reagent strip tests for blood (hemoglobin, myoglobin)
Bromothymol blue turns from yellow to blue which
in urine are based on the peroxidase-like activity of the heme
indicates a pH range 6.0 to 7.6. The strips are available as
portion of hemoglobin molecules. Peroxidase catalyzes the
multiple-reagent strips in combinations with other tests for
release of oxygen from the organic of peroxide in the reagent
urinary constituents. No interferences are known.
strip. The released oxygen reacts with the reduced form of the
chromogen, producing an oxidized chromogen. The reaction
results to a color change which ranges from orange to green
to dark blue. Any green spots or green color development on
the reagent area within 60 secs is significant and the urine
specimen should be examined further.

KETONES (40 SECONDS)

Reagent strip test is based on ketones reacting with
nitroprusside and acetoacetic acid to produce a color change
ranging from light pink (negative result) to darker pink or
purple color (positive results). Ketones are normally not
present in urine. Detectable ketone levels may occur in urine
during physiological stress conditions such as fasting,
pregnancy, and frequent strenuous exercise.

BILIRUBIN (30 SECONDS)

Routine testing for urinary bilirubin by reagent strip is
based on the coupling reaction of bilirubin with diazonium salt
(diazotized dichloroaniline) in a strongly acidic medium. The
reaction produces color ranging from various shades of tan or
tannish purple proportional to bilirubin concentration in urine.

UROBILINOGEN (60 SECONDS

Screening for urobilinogen is useful in the diagnosis of
liver function disorders. Reagent striptest for urobilinogen is
based on a modified Ehrlich reaction between
p-dimethyl-aminobenzaldehyde and urobilinogen in strongly
acidic medium to produce a pink color

NITRITE (60 SECONDS)

The nitrite test is a rapid, indirect method for the
detection of significant & asymptomatic bacteriuria. Reagent
strips for the detection of nitrite in urine depends on the
conversion of nitrate to nitrite by the action of Gram negative
bacteria in urine. In an acidic medium, nitrite in the urine reacts
with p-arsanilic acid to form a diazonium compound. The
diazonium compound then couples with quinoline compound
(1-naphthyl-ethylenediamine) to produce a pink color.
INTERPRETATION OF RESULTS/ CLINICAL
SIGNIFICANCE:
LEUKOCYTE ESTERASE (120 SECONDS)
Neutrophils is the most common leukocyte seen in
urine, and is normally present in low numbers, Neutrophils

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
PROTEIN ● Cystitis
● Pyelonephritis (complication of untreated cystitis)
● Urinary proteins become evident at chemical testing at 30 ● Inflammation of urinary tract
mg/dL. ● Can detect bacteriuria
● However, demonstration of proteins does not always signify ● Rapid screening for Urinary tract infection
Renal disease. Further testing will classify if it’s pathologic or ● Evaluation of antibiotic therapy
not. ● Screening of urine culture specimen
● Pre-renal proteinuria ● Used in combination with LE test
● Renal proteinuria
● Post-renal proteinuria LEUKOCYTE ESTERASE

GLUCOSE This serves as a basis for performance of urine bacterial

culture.
Hyperglycemia – increase in blood glucose. Clinical ● Inflammation of urinary tract
Significance: ● Screening of urine culture specimens
● Diabetes mellitus – impaired function of insulin ● Urinary tract infection (Bacterial/Non-Bacterial)
● Pancreatitis – decreased levels of insulin
● Pancreatic cancer – increased glucagon and somatostatin pH
● Acromegaly - increased growth hormone:
● Cushing syndrome – increased cortisol ● Respiratory or metabolic acidosis/ketosis
● Pheochromocytoma – increased cortisol ● Respiratory or metabolic alkalosis
● CNS damage – increased epinephrine ● Defects in renal tubular secretion and reabsorption of acids
● Stress – increased epinephrine and bases - renal tubular acidosis
● Gestational diabetes – occurs during pregnancy ● Renal calculi formation and prevention
● Treatment of urinary tract infections
BLOOD ● Precipitation / Identification of crystals
● Determination of unsatisfactory specimens
● Hematuria
● Hemoglobinuria SPECIFIC GRAVITY
● Myoglobinuria
● Monitoring patient hydration and dehydration
KETONES ● Loss of renal tubular concentrating ability
● Diabetes insipidus
● Diabetic acidosis ● Determination of unsatisfactory specimens due to low
● Insulin dosage monitoring Concentration
● Starvation
● Malabsorption CLASS ACTIVITY:
● Pancreatic disorders
● Strenuous exercise
● Vomiting
● Inborn errors of metabolism

URINE BILIRUBIN

Detection of B2 can provide early detection of liver disease.

● Hepatic jaundice – the integrity of liver is damaged

■ Hepatitis
■ Liver cirrhosis
■ Other liver disorders (liver cancer)
● Post-hepatic jaundice – Bile duct obstruction
■ Gallstones
■ Pancreatic cancer

URINE UROBILINOGEN

● Liver diseases like hepatitis, carcinoma, cirrhosis

● Hemolytic disorders
● Transfusion reactions
● Sepsis 1. Leukocyte
Esterase
NITRITE

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY

Incubation Time: 120 seconds p-dimethyl

aminobenzaldehy
Principle The esterases de
(Reaction): cleave a and urobilinogen
derivatized in strongly acidic
pyrazole amine medium
acid ester to
liberate a Reagent: p-dimethyl-amino
derivatized benzaldehyde (in
hydroxy pyrazole. strongly acidic
This pyrazole medium)
reacts with
diazonium salt Result: pink (positive)

Result: Beige pink to Clinical Liver disease,

purple (positive) Significance: hemolytic
disorders, liver
Clinical Urinary Tract cancer
Significance: Infection

4. Protein

2. Nitrite Incubation Time: 60 seconds

Incubation Time: 60 seconds Principle Protein error of

(Reaction): “pH Indicators”
Principle Conversion of
(Reaction): nitrate to nitrite Result: Yellow to Green to
by yellow-green Green-blue
the action of (positive) (negative)
Gram negative
bacteria in urine.
Clinical Proteinuria
In an acidic
Significance: (caused by renal
medium, nitrite in
diseases)
the urine reacts
with p- arsanilic
acid to form a
diazonium 5. pH
compound.
Incubation Time: 60 seconds
Reagent: p-arsanilic acid quinoline compound
(forms diazonium Principle double-indicator
compound) (Reaction): system of methyl
red and
Result: pink (positive) bromthymol blue
that
measure urine
Clinical Urinary Tract
pH between pH 5
Significance: Infection, Cystitis
to
9.

Result: (Acidic result) (Alkaline result)

Methyl red- Bromothymol blue-
3. Urobilinogen
red-orange to yellow to blue
yellow (4.4 - 6.2) (6.0-7.6)
Incubation Time: 60 seconds
Clinical Determining
Principle Modified Ehrlich Significance: existence of
(Reaction): reaction between acid-base
disorders

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY

6. Blood 7. Specific
Gravity
Incubation Time: 60 seconds
Incubation Time: 45 seconds
Principle Peroxidase- like
(Reaction): activity of the Reagents: Bromothymol Blue Methyl Red
heme portion of
hemoglobin Result: Alkaline - Acidic -
molecules. Blue-green Yellow-green
alkaline pH acid pH
Result: Orange to green
to dark blue Clinical Proteinuria
(positive) Significance: (caused by renal
diseases)
Clinical Hematuria:
Significance: Renal calculi
Glomerulonephritis
Pyelonephritis
Tumors 8. Ketone
Trauma
Exposure to toxic
Incubation Time: 40 seconds
chemicals
Anticoagulants
Strenuous Principle Ketones reacting
exercise (Reaction): with nitroprusside
and acetoacetic
Hemoglobinuria: acid
Transfusion
reactions Result: Darker pink or Light pink
Hemolytic purple (positive) (negative)
anemias
Severe burns Clinical Diabetic acidosis,
Infections/malaria Significance: Insulin dosage
Strenuous monitoring,
exercise/red Starvation
blood cell trauma
Brown recluse
spiderbites

Myoglobinuria:
9. Bilirubin
Muscular
trauma/crush
syndromes Incubation Time: 30 seconds
Prolonged coma
Convulsions Principle Reaction of
Muscle-wasting (Reaction): bilirubin with
diseases diazonium
Alcoholism/ salt (diazotized
overdose dichloroaniline) in
Drug abuse a strongly acidic
Extensive medium.
exertion
Cholesterol- Result: Tan or Tannish
lowering statin Purple
medications
Clinical Hepatitis,
Significance: Cirrhosis

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
It is one of the most important indicators of renal disease.
Its presence in the urine depends on the nature of the
clinical and pathological disorder and the severity of the
10. Glucose specific disease.
Types of Proteinuria
Incubation Time: 30 seconds 1. Accidental or false proteinuria
2. Physiological or functional proteinuria
Principle Enzymatic reaction 3. Postural (orthostatic) proteinuria
(Reaction): between 4. Renal or true proteinuria.
glucose oxidase,
peroxidase, & Screening Test for Protein in urine: Reagent Strip
chromogen. Method

Result: Green to Brown The reaction is based on the phenomenon known as

the “protein error of pH indicators”
Clinical Hyperglycemia, ❏ The point of color change of some pH indicators is
Significance: Diabetes Mellitus, different in the presence of protein from that observed in
Glycosuria the absence of protein, because proteins act as hydrogen
ion acceptors at a constant pH.
❏ Proteins primarily albumin, when present in urine, are
said to accept hydrogen ions from the indicator.
➢ This will alter the pH of indicators turning
them alkaline and their color will progress from
MODULE 5: Determination of Proteins in Urine green to blue, depending on the amount of
proteins
Introduction ❏ The pad is impregnated with an indicator which is
usually: ➔ Tetrabromophenol blue
Protein is a macromolecule, composed of one or more ➔ Tetrachlorophenol
polypeptide chains, each possessing a characteristic ➔ Tetrabromosulfonphthalein
amino acid sequence and molecular weight. It has many ❏ The development of any green color in the protein
biologically important functions. reagent pad is due to the presence of protein at a
Determination of protein is an integral part of routine constant pH of 3.
urinalysis. Normal urine contains very little protein, usually
❏ Colors range from yellow to yellow-green for negative
1 – 14 mg/dl per 24 hours.
results and green to green-blue for positive results.

Proteins in normal urine consist mostly of plasma
albumin and globulins.
● 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.
Other proteins that originate from the renal tubular cells may
also be present in small amounts in urine. These include:
● Uromodulin (also known as Tamm-Horsfall protein), a
mucoprotein synthesized by the distal tubular cells,
● Urokinase, which is a fibrolytic enzyme, &
● Secretory immunoglobulin A
Clinical Significance
The presence of protein in the urine is called Proteinuria.
Qualitative Tests for Determination of Proteins in
Urine
Presence of an increased amount of protein in urine
(proteinuria) is often the first indicator of renal disease.
For most patients with proteinuria, albumin is the protein
present in increased concentration.
· Qualitative or semiquantitative screening tests for
urine protein rely on protein precipitation techniques.
Proteins denature upon exposure to extremes of pH or
temperature and the most visible evidence is a decrease
in solubility.
The general principle of these tests is that
protein is either precipitated out of the urine specimen by
means of a chemical, which is usually a strong acid, or it
is coagulated out of solution with heat.
Since the result in precipitation tests is
determined by the presence of either turbidity or a
precipitate, it is important that the urine be free from
particles or clear before the test is performed. To clear

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
the urine, it should be filtered or centrifuged. The clear acids. These are not to be reported positive for
filtrate is tested for the presence of protein. protein.
5. The test may be performed by holding a test tube
The amount of turbidity or precipitation is containing a few ml of Robert's Reagent in an
roughly proportional to the amount of protein present in inclined position and allowing the clear urine to
the urine specimen, and the results are generally graded run slowly down the side of the tube from a
as negative, trace, 1+, 2+, 3+, or 4+. pipette.

Note : If bile is present in the specimen, any colors

( red, violet, blue, or green ) will be found at the
line of contact.

Heller’s Ring Test

Principle: Albumin is precipitated by Heller’s reagent (pure

nitric acid) producing white ring, which varies in density with
the amount of albumin present.

Materials:
● Concentrated HNO₃ (in a test tube)
● Urine sample
● Dropper
Procedure:
1. Using a dropper, take a small quantity of urine from the
container.
2. Take the test tube containing concentrated nitric acid.
3. Incline the test tube and add the sample or urine
using a dropper along the inner side of the test tube.
4. As the urine comes in contact with the nitric acid, a
white ring is formed at the point of contact.

Presence of the white ring at the junction of the

two layers indicates the presence of albumin in
the sample.
Robert’s Test
Conclusion:
Principle: Albumin is precipitated by Robert’s reagent Nitric acid causes denaturation of proteins with the
(magnesium sulfate (MgSO4) and nitric acid (HN03)) formation of white precipitate. As urine that contains
producing white ring, which varies in density with the albumin comes in contact with nitric acid, it forms a
amount of albumin present. It is based on the precipitation white ring at the point of contact.
of protein and formation of white compact rings using
concentrated Nitric acid (HNO3). Note: Heller's Test is not suitable for routine
analysis of proteinuria because of the highly
Procedure: corrosive nature of concentrated nitric acid.
1. Place 3-5 ml of clear urine in a test tube.
2. Place the tip of a 5 or 10 ml pipette containing Salt and Acetic Acid Test
Robert's Reagent to the bottom of the tube and
allow 3 ml of the reagent to lay beneath the
urine.
3. A white ring at the zone of contact indicates a
positive test.
4. The ring must be read within 3 minutes after adding
the reagent, and with the eyes on the level of the
contact ring. Rings that are 1-2 mm above the
zone of contact are due to mucin and nuclear
albumin; rings 1-2 cm above the zone of contact
are 60 due to urates, uric acid urea and bile Principle: Albumin is precipitated by heat and coagulated

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
by acetic acid. The amount of precipitate or
cloudiness formed is directly proportional to the amount
of albumin present.

Note:
● When added with reagent, it is necessary to apply
heat until boiling. After 5 minutes, observe for
any turbidity/ cloudiness. It will then form a
precipitate/ coagulation.
● It is considered to be a “coagulation & precipitation
test.”
Video Excerpts:
Aim: To detect the Presence of Albumin in the sample of
Urine by Heat Coagulation Test
Procedure for HEAT and Acetic Acid Test Combined 2
Videos
● Take a test tube, hold it with holder, fill ⅔ of the test
tube with a sample (8mL of Urine- 2nd Video)
● Heat the upper ⅓ of the sample
○ Clinical Use- for bedside detection of
albumin in urine
○ Principle- on application of heat, albumin
gets denatured and coagulum forms at
the upper heated part of the test tube
● Coagulum forms in upper ⅓ part, lower part of the
test tubes serves as control for comparison
● Add 2 drops of Acetic Acid (1 drop of 33% Acetic
Acid 2nd Video) to adjust the pH at isoelectric
point of albumin (4.7).
Turbidity forms, appears, intensifies, or remains as it
is indicates albumin
If turbidity disappears- indicates presence of
phosphates or carbonates
Methods by which Proteins Get Precipitated:
1. Denaturation
2. Dehydration of Outer Shell
3. At its isoelectric Point
4. Neutralization of Charge
Denaturation- loss of secondary, tertiary, and quaternary
structure of proteins, only primary structure remains intact.
Coagulum- thick viscous precipitate
Heat Coagulum test- is used for bedside detection of
albumin in urine in patients with Nephrotic syndrome,
Glomerulonephritis, etc.
Sulfosalicylic Acid Test for Urine Protein
Principle: This precipitation test is based on cold
precipitation of urine protein with a strong acid
(Sulfosalicylic Acid) producing white precipitate.
● Sulfosalicylic acid denatures protein, causing
precipitation.
Note:
● Also known as “Exton`s Sulfosalicylic Test” ●
Can also be spelled as “Sulphosalicylic Test” ● No
need to boil, just add the ragent & heat gently ● It is
considered to be a more specific test compared to
salt acetic test
○ Sulfosalicylic reagent is a combination of
Anhydrous Sodium Sulfate &
Sulfosalicylic Acid Crystals
○ Anhydrous Sodium - serves as a
precipitant of protein (precipitates without the need of
boiling) ● However, not used routinely because it is
expensive.
● Although specific, salt acetic test is more preferable
as routine confirmatory test.
Video Excerpts:
Materials:
● Urine sample
● 30 % Sulphosalicylic Acid
● Dropper
Procedure:
1. Using a dropper, take a small quantity of
Sulphosalicylic acid.
2. Add it to the test tube containing the urine
sample. 3. A Cloudy/ Turbid solution or
Precipitate of Protein in the sample indicates
presence of Albumin.
Conclusion:
When sulphosalicylic acid reacts with albumin, it
denatures the protein in the sample that appears in the
form of precipitate.
Test for Bence Jones Protein (BJP) in Urine: Harrison’s
Method (Heat & Acetic Acid Test)
Bence Jones protein (BJP) is a low molecular weight protein
that is excreted by persons with proliferative disorder like
multiple myeloma, a disorder of immunoglobulin producing
plasma cells. This protein is filtered in quantities exceeding the

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
tubular reabsorption capacity and is excreted in the urine.
However not all persons with multiple myeloma excrete
detectable levels of Bence jones protein. Serum
electrophoresis is the method of choice for diagnosing
suspected cases of multiple myeloma to confirm screening
method by urinalysis.
Most of the screening tests are based on precipitation of
BJP at temperature between 40-60°C and solubility at
100°C. Other tests are based on its precipitation at cold
temperature with salts, ammonium sulfate and acids.
Principle:
❏ This screening test is based on the unique
solubility characteristics of Bence Jones protein.
❏ This simple screening test is done by gradually
heating the urine up to the boiling point.
❏ Unlike other proteins which coagulate and remain
coagulated when exposed to heat, it coagulates
at temperatures between 40°C to 60°C and
dissolves when the temperature reaches 100°C.
❏ A specimen which is turbid between 40°C to 60°C
and clear at 100°C is suspected of containing the
protein.
Video excerpts:
● What does a positive Bence Jones test mean? -
One of the several tests used to diagnose a
type of blood cancer known as multiple
myeloma. Between 50 and 80 percent of
people with MM will have Bence Jones
protein in their urine. It is also linked to
cancers of the lymphatic system .
out the presence of Bence Jones protein.
Qualitative Test for Determination of Proteins in
Urine: Esbach’s Test
The presence of protein in the urine, (proteinuria), in routine
urinalysis does not always signify renal disease; however, its
presence does require additional testing to determine whether
the protein represents a normal or a pathologic condition.
Hence, quantitative determination of proteins in the urine
should be performed. Clinical proteinuria is indicated at ≥30
mg/dL (300 mg/L).
Principle: Proteins in urine even in the smallest (trace)
amount reacts with and are precipitated by picric acid.
The amount of precipitated protein is measured in g/L.
Video excerpts:
Principle: Eashbach’s method of protein precipitation in
which picric acid is used to precipitate the protein and
amount of protein precipitate is measured by volume.
Albuminometer:
● Thick walled glass tube graduated in grams of dried
albumin per 1000mL of urine
● Two markings are seen “U” & “R”
➔ “U” - Urine
➔ “R” - Reagent

Test requirements:
● 3% Acetic acid
● Concentrated HCL
● Urine sample
● Test tubes

Test procedure:
Uses of Esbach’s Albuminometer
1. Add 1-2 drops of 3% acetic acid to the urine
sample to bring it to acidic pH ● Quantitative estimation of albumin in urine
2. Centrifuge the urine sample 3000 rpm for 5 minutes.
3. Add 1 mL of concentrated HCL to a test tube Layer Components of Esbach’s Reagents:
1 mL of centrifuged urine sample slowly along the
wall of the test tube and observe the interface. ● Picric acid – 10 gm – (precipitate albumin)
● Citric acid – 20 gm – (dissolved phosphate)
Interpretation:
● Distilled water – 1000 mL
● If Bence Jones protein is present, it will be Note: If there is a presence of phosphate in the urine, the
precipitated by the HCL and will form a fine or measurement of albumin will be inaccurate. Thus the
heavy ring at the interface of urine and HCL. phosphate must be dissolved.
● A positive test should be confirmed by the heating
test because occasionally albumin may also Sample Preparation
precipitate. ● The absence of a ring definitely rules

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
● Samples should be 24hrs urine day) Causes:
● Should be clear by filtering and acidifying 10%
acetic acid ❏ Post streptococcal glomerulonephritis
● If specific gravity > 1.025, it should be diluted 1:2 or ❏ Primary renal disease
Table .2 Proteins in Urine
1:4, otherwise albumin precipitate will float

Procedure

1. Fill with filtered acidified urine of correct specific

gravity up to the “U” mark
2. Fill with the Esbachs’s Reagents up to “R”
mark 3. Stopper the tube
4. Mix the contents well
5. Keep this tube in a standing position for 24 hours 6.
Look at the precipitation after 24 hours and record the
results

Recording of results

● Albumin is expressed in g /L of urine

● The results should be divided by 10 to get g % ● If
the urine is diluted, multiply the results by dilution
factors
● Thus 24 hrs excretion = x / 10 g %
➔ Where x is the amount of albumin
precipitate ● Normal value is 100 to 150 mg/ 24 CLASS ACTIVITY:
hrs
A. Robert's Test Principle: Albumin is precipitated
Interpretation: Albuminuria by Robert’s Reagent producing
white ring, which varies in density
1. Asymptomatic (<3mg albumin excreted per with the amount of albumin
present
day) Causes: Reagents used: magnesium
sulfate and Nitric acid (HNO3)
❏ Excessive exercise (MgSO4) / Robert’s Reagent
❏ Pregnancy Visible positive result: Albumin is
❏ Orthostatic albuminuria present, white ring will be formed.

2. Mild Proteinuria (<1.0 g albumin excreted per

B. Principle:
day) Causes: Reagents used:

❏ Hypertension Visible positive result:

❏ Polycystic kidney
❏ Chronic nephritis
❏ Fever
❏ UTI

3. Moderate Proteinuria ( 1 – 3 g albumin excreted per

day) Causes:

❏ Chronic glomerulonephritis
❏ Nephrosclerosis
❏ Multiple myeloma

4. Massive Proteinuria (>3 g albumin excreted per

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
Principle:
Reagents used:
Visible positive result:
Principle:
Reagents used:
Visible positive result:
Principle: This precipitation test is
based on cold precipitation of
urine protein with a strong acid.
Reagents used: 7% Sulfosalicylic
Acid Reagent
Visible positive result: Cloudy
Turbid Solution
MODULE 6:Test for Glucose (Dextrose) in Urine
Introduction
Glucose is the sugar commonly found in the urine. The
presence of glucose in the urine is termed glucosuria
/glycosuria. Renal glucosuria occurs with normal blood
glucose levels because tubular reabsorption of glucose is
below normal, thus permitting some glucose to spill into
the urine.
Glucosuria is caused by:
● prerenal condition (hyperglycemia
● a renal condition (defective tubular absorption)
There are two basic types of tests that are used to screen
for or monitor glycosuria. The procedures that use the
enzyme glucose oxidase are specific for glucose, while the
copper reduction tests will detect any reducing substance.
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As with all screening procedures, a positive test result
should be correlated with other findings.
Routine urinalysis includes a test for glucose (dextrose).
The test for glucose is routinely performed on urine
sample. It is used to diagnose and monitor a metabolic
disorder. The presence of glucose in urine (glycosuria or
glucosuria) indicates that the metabolic disorder diabetes
mellitus should be suspected, although several other
conditions may yield positive results
Physiologically glycosuria is observed after intake of large
amounts of sugar or foods containing sugars had been
eaten, during acute emotional stress, I.V. infusion of fluids
containing glucose, and after exercise. It may also be
associated with pregnancy, certain types of meningitis,
endocrine disorders like hypothyroidism & certain tumors
of the adrenal glands, increased intercranial pressure, &
some brain injuries. The occurrence of measurable
glucose in the urine is not normal because under
conditions, all the glucose in blood is filtered by the
glomerulus and reabsorbed into the blood circulation.
Principle of Benedict’s Test
This test is based on the ability of glucose and other
substances to reduce copper sulfate (CuSO4) to cuprous
oxide (Cu2O) in the presence of alkali and heat. When the
reaction takes place, a color change occurs ranging from a
negative blue (CuSO4) through green, yellow, to red
orange (Cu2O), depending on the amount of glucose
present in the urine. This test will detect 0.15% to 0.2% of
glucose. Benedict’s solution is not reduced by uric acid,
creatinine, chloroform or aldehyde. This test is not specific
for glucose and is affected by most reducing substance if
they occur in large quantities.
Procedure:
1. Prepare a clean dry test tube, label it properly.
2. Pipette 5 ml of Benedict’s reagent into it.
3. Add 8 drops (gtt.) of urine.
4. Mix by shaking and place the tube in vigorously
boiling water for 5 minutes.
5. Remove the tube from the boiling water and read
result at once.
6. In the presence of glucose the entire solution will
be filled with precipitate ranging in color from
yellowish to green to orange to brick red or red
orange.
22
 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
■ Clinistix – O-toluidine chromogen color changes
from pink to purple.
Interpretation of Results ■ Multistix – Potassium iodide chromogen color
changes from blue to brown.
■ Chemstrip – Aminopropy-carbazol chromogen
color changes from yellow to orange brown.
■ Testape – Orthotolidine color changes from
yellow to blue.

Reagent Strip for Glucose Test

Grade Precipitate Supernatant Mixed

Solution

Negative none No change No change

in blue color in blue color

Trace small blue blue green

amount of
yellow ppt

1+ moderate blue apple green

amount of
yellow ppt

2+ large blue muddy

amount of green/yellow
yellow ppt

3+ large light blue muddy

amount of orange
yellow ppt

4+ large colorless, orange to

amount of slight yellow red
yellow to red
ppt

Methods used in Glucose

Strip Method

enzymatic, specific & sensitive, cost effective, noninvasive

means. Glucose reagent strip was the first “dip & read”
reagent strip developed by Miles, Inc. in 1950. Glucose
oxidase impregnated on the reaction pad rapidly catalyzes
the oxidation of glucose to form hydrogen peroxide &
gluconic acid. The hydrogen peroxide formed oxidizes the
chromogen on the pad in the presence of peroxidase. The
color change observed depends on the chromogen
(substances used in chemical testing requiring the
development of color during the reaction) utilized in some
common dipstick tests that is included in the following:

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY

CLASS ACTIVITY
Copper Reduction Test
I. BENEDICT’S TEST

Principle? This test is based on the ability of glucose

and other substances to reduce copper
sulfate (CuSO4) to cuprous oxide (Cu2O) in
the presence of alkali and heat. When the
reaction takes place, a color change occurs
ranging from a negative blue (CuSO4)
through green, yellow, to red orange (Cu2O),
depending on the amount of glucose present
in the urine. This test will detect 0.15% to
0.2% of glucose. Benedict’s solution is not
reduced by uric acid, creatinine, chloroform
or aldehyde. This test is not specific for
glucose and is affected by most reducing
substance if they occur in large quantities.

Reagents Benedict’s Reagent

used?

Amount of 5 mL
reagent
Clinitest Tablet used?

The principle of clinitest is essentially the same as that of Amount of 8-10 drops of urine
Benedict’s Qualitative Test. The clinitest tablet may be urine
thought of a solid form of Benedict’s reagent. It promotes used?
“effervescence”
Number of 5 minutes
minutes in
boiling?

Reasons Presence of other reducing sugars, peroxide

for False or strong oxidizing agents (detergents,
Positive? formalin), drugs (levodopa, penicillin,
streptomycin, salicylates, oxytetracycline,
ascorbic acid, homogentistic acid, dextran
p-aminosalicylic acid, vitamin c,
glucuronides)

II. IDENTIFY THE GRADING OF THE FOLLOWING

RESULTS

Visible result: Blue

colored solution

Grading: None (Negative)

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY

Visible result: Green Visible result: Apple-Green

colored solution colored solution
Blue with yellow ppt
Grading: Traces of
Grading: Traces of Reducing Sugar (2+)
Reducing Sugar (1+)

Visible result: Orange

colored solution
Visible result: Yellow
Grading: Large amount of colored solution
reducing sugar (3+)
Grading: Traces of
Reducing Sugar (2+)

Visible result: Red-Orange

colored solution

Grading: Large amount of

reducing sugar (4+)

III. GIVE THE PRINCIPLE OF THE FOLLOWING

TESTS FOR GLUCOSE

Visible result: Brick-Red Clinitest Tablet ● Copper Reduction Test

colored solution ● The tablet contains copper sulfate,
sodium carbonate, sodium citrate
Grading: Large amount of and heating hydroxide. Upon
reducing sugar (4+) addition to the water and urine, heat
is produced by hydrolysis of sodium
hydroxide with its reactant sodium
citrate and carbon dioxide is
released from the sodium carbonate
to prevent air from interfering with
Visible result: Yellow- the reduction reaction.
Green colored solution
Reagent Strip ● Glucose Oxidase Test
Grading: Traces of ● Glucose oxidase catalyzes the
Reducing Sugar (2+) reaction between the glucose and
oxygen or the air to produce
gluconic acid and peroxide. The
peroxidase catalyzes the reaction
between peroxide and chromogen
Visible result: Red-Orange
forming an oxidized colored
colored solution
compound that is directly
proportional to the glucose
Grading: Large amount of
concentration.
reducing sugar (4+)

IV. GIVE THE EXPECTED RESULTS IN BENEDICT'S

Visible result: Blue
TEST OF THE FOLLOWING CONDITIONS
colored solution

Grading: None (Negative) Patient suffering from Negative - diabetes insipidus

diabetes insipidus means that the sugar is not
increased.

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
Urine from a patient who Positive - it consumes
just had unlimited buffet carbohydrate and it is
converted into glucose.
Excess glucose in blood will
be excreted in urine.
Patient suffering from Negative - urinalysis is
urinary tract infection indicative for UTI not for
glucose.
Patient suffering from Positive - Epinephrine is
severe stress increased which also
increases the activity of the
body and by that ,release of
glucose is also increased.
Patient with Fanconi Positive - Blood glucose is
syndrome increased by fanconi
syndrome which affects the
ability of the kidney to absorb
all substances (tubular
reabsorption is defective)
Patient with acute Positive - Pancreas is
pancreatitis damaged; less insulin
produced → high blood sugar
MODULE 7: DETECTION OF KETONE BODIES IN URINE
INTRODUCTION
Normally, the end products of fatty acid metabolism
are carbon dioxide and water. No measurable ketones are
produced. However, when carbohydrate availability is limited,
the liver must oxidize fatty acids as its main metabolic
substrate. As a result, large amounts of acetyl coenzyme A are
formed and the Krebs cycle becomes overwhelmed. To handle
the increased acetyl coenzyme A load, the liver mitochondria
begin active ketogenesis. Large quantities of ketones are
released into the bloodstream (ketonemia) and provide energy
to the brain, heart, skeletal muscles and kidneys. The amount
of each ketone body in the blood varies with the severity of the
condition. The average distribution of ketones in serum & urine
is:
■ 78% Beta-hydroxybutyrate
■ 20% Acetoacetate
■ 2% Acetone
- products of incomplete fat metabolism
- not found in urine under normal conditions because
fats are completely metabolized to form carbon
dioxide and water.
-
Increased fat metabolism takes place when there is inability to
metabolize carbohydrates as found in diabetes mellitus;
increase loss of carbohydrate from vomiting; inadequate intake
of carbohydrate associated with starvation and malabsorption.
Testing for ketone bodies in urine is medically relevant
in the management and monitoring of insulin dependent
diabetes mellitus type 1 (IDDM1). The presence of ketones in
the urine may be indicative of acidosis. Ketonemia (ketones in
the bloodstream) and ketonuria (ketones in urine) are seen in
uncontrolled diabetes mellitus. In diabetic ketonuria, presence
of ketoacidosis may provide a warning of impending coma.
Urine tests for ketone are commonly accompanied
tests for glucose. Different tests measure acetoacetic or both
acetone and acetoacetic acid. Ketones are sensitive and even
preservatives cannot prevent deterioration of ketones in the
urine. Bacterial degradative action causes the loss of
acetoacetic acid in In vitro and in vivo. Thus, acetone should
be preserved by keeping urine in a closed container inside the
refrigerator.
PROCEDURES
ROTHERA’S TEST
● Principle:
Acetone chemically reacts with alkaline
nitroprusside to give a purple color. This property has
become the basis of its detection in urine samples
Note : Reddish-purple ring at the zone of contact appearing
within ½ minutes indicates a positive result while brown ring
indicates a negative result.
Result Interpretation
No ring or brown ring Negative
Faint pinkish-purple ring Trace
Distinct pinkish-purple ring 1+

The search for a more specific & sensitive method for Narrow dark purple ring 2+
ketone detection resulted in the nitroprusside test, which was
originally developed by Legal in 1883 and was later modified Wide dark purple ring 3+
by Rothera's in 1908. Although the test is no longer performed (appearing very rapidly)
routinely in clinical laboratories. Rothera's tube test is more
sensitive to acetoacetate & acetone than the current test
modification available on reagent strip or tablet tests. GERHARDT’S TEST
● Medically relevant in the management of diabetes
mellitus type I. ● Principle:
Gerhardt’s test depends upon the reaction
Ketone bodies between aceto-acetic acid and ferric chloride to form
- such as: Acetone, diacetic acid (aceto-acetic acid), bordeaux red compound. Certain drugs such as
Hydroxybutyric acid (78%) are intermediary salicylates interfere with the test and give a false
products of fat metabolism. positive reaction.

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 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY

Note: If acetoacetic acid was present, the test will now

become negative.If interfering substances are present, the test Interpretation Clinical Significance
will remain positive.
Positive Insufficient carbohydrate intake
HART’S TEST
● Principle:
Beta-hydroxybutyrate and acetoacetic acid
acid. It comprises 78% of the total ketones in urine.
Beta-hydroxybutyric and acetoacetic acid contribute
excess hydrogen ions to blood, resulting in acidosis.
Acidosis is an extremely serious condition and results
in death if allowed to continue. the removal of acetone
and acetoacetic acid by boiling and the reaction of the
remaining beta-hydroxybutyric acid with hydrogen
peroxide constitute the Hart’s Test.

Note: A red ring at the zone of contact will be seen in the tube
containing hydrogen peroxide if beta-hydroxybutyric acid is Experiencing polyuria, Blood glucose results is 4++++
present, while no reaction will be detected on the other tube.

CLASS ACTIVITY
Interpretation Clinical Significance
EXPLAIN THE KETONURIA IN THE FOLLOWING CASE
STUDIES Positive ● Deficiency of Insulin → High blood sugar
● High blood sugar → Diabetes
(Polyphagia, Polyuria)
○ Diabetes → Insufficient
carbohydrate intake
○ Polyuria → Dehydration

Having loose bowel movement for 48 hours

Interpretation Clinical Significance

WEIGHT LOSS PROGRAM DAILY. TWICE A DAY GOING TO
Positive Dehydration (loss of water, carbohydrate THE GYM. DIET PLAN
is absent → Faulty metabolism of fats)

Interpretation Clinical Significance

Positive ● Insufficient carbohydrate intake

● Dehydration

Employees having labor problems, so they decided to

hunger strike

#RMT2024 BUENAVISTA, GABUNAS, ISUGON, PAMOTILLO || BSMLS 3-B 27

 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
Child having a very high fever, experiencing delirium, has
no appetite
Interpretation Clinical Significance
Positive ● Dehydration → No food intake
(carbohydrate is absent, stored
energy is depleted)
● Body heat absorbs fluid →
Dehydration
MODULE 8: DETECTION OF BILE PIGMENTS IN URINE
Bilirubin is an intensely ​orange-yellow pigment that
when present in significant amounts causes a characteristic
coloration of plasma & urine. The primary source of bilirubin
(85%) is hemoglobin released daily from the breakdown of
senescent (old/aging) RBCs in the reticuloendothelial cells of
the ​spleen & bone marrow​.
Three Major Types of Jaundice
Since these types differ in the substances excreted in the
urine, they can be differentiated by testing for the presence of
bilirubin & urobilinogen.
Hemolytic (Prehepatic)
Type of jaundice which is the result of excessive
production of bilirubin. The increased breakdown of RBCs
produces bilirubin at a rate exceeding the ability of the liver to
conjugate & excrete it. Liver function is still normal.
● Possible causes include: intravascular hemolysis/
transfusion reactions, hemolytic anemia, hereditary
spherocytosis, sickle cell disease.
Hepatic (Hepatocellular)
Results from liver damage, altered bilirubin
mechanism is hepatic in origin & results from hepatocellular
disease or disorders.
● Clinical picture varies according to the type & degree
of hepatic injury. There may be injury to the
parenchymal cells caused by viral hepatitis or
cirrhosis, genetic disorders, intrahepatic disease as a
result of chemical intoxication or drug reactions.
#RMT2024 BUENAVISTA, GABUNAS, ISUGON, PAMOTILLO || BSMLS 3-B
Obstructive (Post Hepatic)
It is due to an obstruction in the common bile duct
or biliary system
● causes includes: gallstones, carcinoma/tumors,
pancreatitis, fibrosis, diseased lymph nodes
surrounding the duct, or by carcinoma of the head of
the pancreas.
The appearance of bilirubin in the urine can provide
an early indication of liver disease. It is often detected long
before the development of Jaundice. Appearance of
conjugated bilirubin in urine is caused when the normal
degradation cycle is disrupted by bile duct obstruction or by
liver damage, allowing leakage of bilirubin into the circulation.
Detection of bile pigments can be done by the use of reagent
strip, foam test, iodine test, spot test, tablet test and others.
Bile Pigment Detection Tests
Foam Test
Principle:
Bile pigments give a greenish to yellow, yellow or
brown colored foam that persist for several minutes when
shaken vigorously. It is not a very accurate test because
proteins can also form foam.
1. Obtain two 10 mL test tubes.
2. Pour about 5mL of the well mixed urine that
is to be tested into one test tube.
3. Pour about 5mL of normal urine into the
other tube. This will be used for color
comparison.
4. Cover both tubes and shake. Note the color
of the foam.
5. If the foam has a yellow color that persists
for several minutes, bile may be present and
confirmatory tests should be done.
Note: Certain drugs such as pyridium, serenium,
acriflavine and urobilin compounds also give a yellow
foam, so it is not a specific and sensitive test for bile.
6. Record and interpret the result correctly
Interpretation Result
NEGATIVE Faint yellowish to brown foam
POSITIVE Green foam
1+ Pale green foam
2+ Intense green foam
Smith’s (Iodine) Test
Principle:
When iodine (Lugol’s Iodine or tincture of iodine) is
added to urine containing bile pigments, a green color is
produced.
28
 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
1. Measure 10 mL of urine in a test tube.
2. Dilute 1 mL tincture of iodine with 9 mL of Foam Test
95% alcohol.
3. Overlay the urine with the diluted tincture of
iodine.
Note: An emerald green ring at the zone of contact shows
the presence of bile pigments.
4. Record and interpret the result correctly.
(Refer to the table above for the grading of
results)

Gmelin’s Test
Principle:
When nitric acid is added to urine containing bile
pigments, a play of colors such as green and violet appear at
the zone of contact.
1. Measure 10 mL of urine Smith’s Test
2. Add a few drops (2-4 drops) of slightly yellow nitric
acid using the contact method
3. Record and interpret the result correctly

Note: A play of colors, chiefly green and violet, denotes

the presence of bile pigments. Indican and urobilin
produce blue and red colors. Indican gives a violet color.

Harrison Spot Test

Principle:
After precipitation of the bile pigments by barium
chloride (BaCl2), it is oxidized by acids to derivatives such as
biliverdin.
1. Pour about 20 mL of urine into a small test flask.
2. Add 10 mL of 10% barium chloride
3. Mix and let stand for a few minutes.
4. Filter
Fouchet’s Test
5. Place a few drops of Fouchet’s reagent on the filter
paper
6. A blue to green color is positive for bile.
7. Record and interpret the result correctly

Interpretation:

Physical Examination

Gmelin’s Test

#RMT2024 BUENAVISTA, GABUNAS, ISUGON, PAMOTILLO || BSMLS 3-B 29

 ANALYSIS OF URINE AND BODY FLUIDS LABORATORY
5. DIFFERENTIATE THE THREE TYPES OF JAUNDICE AND
Class Activity WHAT ARE THE FINDINGS IF URINE UROBILINOGEN AND
1. PRECAUTIONARY MEASURES DONE AT HOME IF ONE BILIRUBIN WERE PERFORMED?
OF THE FAMILY MEMBER IS SICK WITH HEPATITIS A
VIRUS.
1. Hemolytic - this type of jaundice is due to excessive
- have yourself vaccinated. production of bilirubin. The increased breakdown of
- avoid using the same utensils. RBCs produces bilirubin at a rate exceeds the ability
- observe proper hygiene by washing hands regularly. of the liver to conjugate & excrete it.
- always clean the bathroom everyday by carefully ● Bilirubin - normal / negative
washing the sink and toilet with disinfectant containing ● Urobilinogen - positive
bleach. 2. Hepatic - results from liver damage, altered bilirubin
- Make sure to flush the urine and feces down the toilet. mechanism is hepatic in origin and results from
Next, have good nutrition and rest by eating a hepatocellular disease or disorders.
well-balanced diet. ● Bilirubin - positive
- have proper medical care for people who are infected. ● Urobilinogen - increased
3. Hepatic - results from liver damage, altered bilirubin
2. FOODS THAT MAY BE AVOIDED IF YOU HAVE mechanism is hepatic in origin and results from
HEPATITIS. hepatocellular disease or disorders.
● Bilirubin - positive
● Urobilinogen - normal
- Meat
- Fried foods / oily foods
- Alcoholic beverages
- Highly processed foods
- Salty foods

3. WHAT ARE THE BLOOD CHEMISTRY TESTS IF

POSITIVE FOR URINE BILE?

● Liver function tests

○ SGPT - highly elevated
○ SGOT
○ ALP - Alkaline phosphatase - increase bone
cell
○ AST
○ FBS
○ Lipid profile REFERENCES
● Bilirubin test for B1 and B2 - most specific for bilirubin
○ B1 unconjugated bilirubin / indirect Notes from the discussion by: Buenavista, Gabunas,
■ Bound to albumin Isugon, Pamotillo
○ B2 conjugated bilirubin / direct
■ Conjugated to glucoronic acid
■ Bilirubin glucocornide Lecturer: Alyssa Lagon
● Serology test
○ Hepatitis viirus panel - to test for positive University of San Agustin PowerPoint presentation: AUBF
infection Lab manual
● Screening
○ HEPATITIS B antigen- preliminary test

4. PROPER STORAGE AND COLLECTION OF SPECIMEN

FOR BILE PIGMENTS.

- Protect from light

- Refrigerate sample
- Use dark colored / amber bottles
- Careful blood extraction to avoid hemolysis

#RMT2024 BUENAVISTA, GABUNAS, ISUGON, PAMOTILLO || BSMLS 3-B 30