Chapter I: INTRODUCTION TO CLINICAL CHEMISTRY

Clinical Laboratory: a facility where tests are done on specimens from the human body to obtain
information about the health status of a patient for the prevention, diagnosis and treatment of diseases

PURPOSES OF THE LABORATORY : gives information to the physician to:

1. detect disease or predisposition to disease
2. confirm or reject a diagnosis
3. establish prognosis
4. guide patient management
5. monitor efficacy of therapy

FACTORS CONTRIBUTING TO THE GENERATION OF QUALITY LABORATORY RESULTS:

1. purity of reagent solutes and solvents
2. quality of containers
3. reliability and quality measuring devices and methods
4. appropriate choice of separative methods and devices
5. observance of established safety procedures
6. understanding of the chemical reaction involved in each test and the effect of physical variables on the
procedure

CLINICAL CHEMISTRY:

a. Fundamental science – seeks to understand the physiologic and biochemical processes occurring in
normal and abnormal states
b. Applied science – analyses performed on body fluids or tissues to provide important information for the
diagnosis and treatment of disease

LABORATORY REAGENTS:

A. CHEMICALS: Analytic chemicals exist in varying grades of purity

1. Analytical Reagent Grade / Reagent Grade Chemicals

✓ meet specifications set by the American Chemical Society
✓ of high purity and is suitable for most analytical laboratory
✓ Include chemicals such as: spectograde, nanograde and HPLC grade

2 FORMS:
A. Lot-Analyzed Reagents- each individual lot is analyzed and the actual amount of impurity is
reported.
B. Maximum Impurities Reagents- the maximum impurities are listed.

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2. United States Pharmacopoeia (USP) and National Formulary (NF)
✓ used to manufacture drugs
✓ not pure enough for use in most chemical procedures.

3. Chemically pure (CP) / Pure Grade Chemicals

✓ Less pure grade, not recommended for reagent preparations.
✓ Impurity limitations and chemical preparation are not uniform

1. Technical or Commercial grade

✓ lowest quality and should not be used for analytical work
✓ Primarily used in manufacturing

B. STANDARDS

1. Primary Standard (PS)

✓ highly purified chemicals that can be measured directly to produce a substance of exact known
concentration.
✓ used for standardization of solutions of unknown strength
✓ stable and can be dried, preferably at 104 - 110˚C, without a change in composition;
✓ not hygroscopic
✓ IUPAC requires PS to be at least 99.98% pure; working standard be 99.95%

2. Secondary Standard
✓ of lower purity with concentration determined by comparison with a primary standard
✓ concentrations cannot be exactly known by direct measurement

3. Standard Reference Materials (SRM)

✓ certified by the National Bureau of Standards (NBS); used as primary standard materials in the clinical laboratory.
✓ Is often used to verify calibration or accuracy/bias assessments.

C. WATER SPECIFICATIONS

REAGENT GRADE WATER: Is a water suitable for reagent and standard preparation.

Types of Reagent Grade Water:

1. Distilled Water: is purified to remove almost all organic materials.
2. Deionized Water: is produced from distilled water using either an anion or cation exchange resin followed
by replacement of the removed particles with hydroxyl or hydrogen ions respectively.

CAP( College of American Pathologists) and NCCLS ( National Committee for Clinical Laboratory Standards)
specifications for Reagent Grade Water:
Characteristics Type I Type II Type III
O
Resistivity (megaohm/cm (@ 25 C) 10 2.0 0.1
Silicate (mg/L, as SiO2) 0.05 0.1 1.0
pH NS NS 5-8
Microbiologic content (CFU/mL) <10 103 NS

A. Type I
➢ Used in test methods requiring minimum interference and maximum precision and accuracy
 For trace metal analyses , iron and enzyme analyses, electrolyte measurements, tissue or cell
culture , Ultra-micro chemical analysis, and preparation of all standards

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 B. Type II
➢ acceptable for most analytic procedures
 chemistry, hematology, immunology, reagent QC & standard preparation
 stored in a manner that reduces any chemical or bacterial contamination and for short periods.
C. Type III
 Used for most qualitative measurements/examinations.
 Used in urinalysis, parasitology, histology, acceptable for washing glasswares and
procedures not requiring Type I or Type I water.

PREPARATION OF REAGENT GRADE WATER

1. Distillation
➢ Water is boiled and vaporized and condensed to remove impurities .
➢ Some impurities include sodium, potassium, manganese, carbonates and sulfates.
➢ Oldest method of water purification.

2. Filtration
➢ remove 98% of the particulate matter.
➢ Filtration cartridges are composed of glass, cotton, activated charcoal which removes organic
materials and chlorine
➢ Submicron filters (<0.2 mm): removes bacteria
➢ Ultrafiltration and Nanofiltration : Remove particulate matters, microorganisms, pyrogens &
endotoxins

3. Deionization
➢ Uses an anion or cation exchange resin followed by replacement of the removed ions with OH- or
H+.

4. Reverse Osmosis
➢ Uses pressure to force water through a semipermeable membrane that acts as a molecular filter.
➢ Does not remove dissolved gases; may be used as pre-treatment of water.

5. Ultraviolet oxidation; Ozone treatment

➢ The use of UV radiation at the biocidal wavelength of 254nm eliminates many bacteria and cleaves
many ionizing organics that are then removed by deionization.
➢ Also removes some trace organic materials

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LABORATORY GLASSWARES

FACTORS DETERMINING THERMAL DURABILITY:

1. Strain Point – temperature resulting to deformation due to heat stress
2. Annealing Point - °T at w/c glass is heated in order to prevent brittleness
3. Coefficient of Expansion – refers to dimension change w/ °T (ideally, it should be low)

PROPERTIES OF GLASS:
a. Breakability: dependent on silicate anion content (greater amount – more durable)
b. Thermal Durability: dependent on boron oxide, nickel & ferric ion content
c. Transparency: dependent on ferric ion content

TYPES OF GLASS:

1. HIGH THERMAL RESISTANCE

A. Borosilicate w/ Low Alkaline content

✓ With high degree of thermal resistance ( 510OC)
✓ This should not be heated beyond its strain point
✓ May cloud/etch when used with strong alkalis ; may be scratched
✓ Most common type used in volume measurements
✓ Most common type used in volume measurements

B. Alumina-silicate glasswares
✓ Corex
✓ Strengthened chemically rather than thermally; 6X stronger than borosilicate glass but less thermally resistant
✓ Alkali resistant; Resists some clouding and scratching
✓ Vycor- ashing & ignition techniques; can withstand very high temperature. Heatable to 900⁰C and can withstand
downshock from 900⁰C to ice water.

2. HIGH SILICA GLASSWARES (96% silica)

✓ with good thermal endurance (900 - 1200OC), chemical stability and electrical characteristics
✓ Radiation-resistant
✓ Ideal for high precision analytical work
✓ good optical and temperature characteristics

3. BORON-FREE GLASS (“Soft Glasswares”)

✓ With poor heat resistance but has high resistance to alkali

4. LOW-ACTINIC GLASS
✓ Thermally resistant and with a red or amber color
✓ Highly protective for handling heat-labile substances in the 300-500 nm range

5. FLINT GLASS
✓ soda lime glass composed of a mixture of oxides of Silicon, Calcium and Sodium
✓ cheapest and with poor resistance to high temperatures

❖ Class A tolerances according to NIST : high thermal borosilicate or aluminosilicate glass ( Preferred
for laboratory applications)

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SPECIAL GLASSWARES:

1. Colored and Opal Glasses

✓ has metallic oxides; used for filters and light bulbs

2. Coated Glass
✓ has a thin, metallic oxide permanently fire-bonded to its surface; can conduct electricity

3. Optical Glass
✓ made of soda lime, lead and borosilicate
✓ has a high optical activity; prisms, lenses and optical mirrors

4. Glass Ceramics
✓ with high thermal resistance, chemical stability and corrosion resistance
✓ for hot plates, table tops and heat exchangers

5. Radiation-Absorbing Glass
✓ made of soda lime and lead

LABORATORY PLASTIC WARES

1. Polystyrene (PS)
✓ Clear and rigid; not autoclavable
✓ Used for disposable wares
✓ Not recommended for use with acids, aldehydes, ketones, ethers, hydrocarbons or essential oils.
✓ Alcohols and bases can be used but not to be stored longer than 24 hours.

2. Polyolefins (polyethylene & polypropylene): unique group of resins with relatively inert chemical
properties.

A. Unaffected by acids Polyethylene

✓ Chemically resistant to most substances except for aldehydes, amines, ethers, hydrocarbons and essential
oils
✓ Conventional Polyethylene (CPE): translucent and flexible; not autoclavable; more expensive

B. Polypropylene (PP)
✓ Has the same chemical resistant as polyethylene
✓ Translucent and rigid; autoclavable but absorbs pigment and tends to become discolored.
✓ Used for screw-cap closures

3. Teflon fluorocarbon resins

✓ Resin that has excellent chemical resistance to almost all chemicals in the lab.
✓ Clear, translucent and flexible; autoclavable
✓ Used for stopcocks, wash bottles and tubings, for cryogenic experiments and work at high
temperatures over extended periods ( -270℃to 255℃)

4. Tygon
✓ Translucent and flexible; nontoxic, clear plastic of modified PVC ( polyvinylchloride), autoclavable;
✓ used for tubings

5. Polycarbonate (PC)
✓ Very susceptible to damage by most chemicals. Resistant to water, aqueous salts and inorganic acids for a long
period.
✓ Twice as strong as polypropylene ( from -100⁰C to 160⁰C)
✓ Very clear and rigid; autoclavable
✓ Used for carboys , ideal for centrifuge tubes and graduated cylinders.

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 6. Polyvinyl Chloride (PVC)
✓ Used for most bottles and tubings

DISADVANTAGES OF PLASTICWARES:
1. Evaporation of solutions – increase in concentration
2. Absorb dyes/pigments – decreased reaction accuracy
3. Color is difficult to describe

STERILIZATION OF HIGH QUALITY PLASTICWARE:

1. Autoclaving: 121°C at 15 psi for 15-20 mins

2. Chemical sterilization: Benzalkonium chloride
3. Gas sterilization: Ethylene oxide

CLEANING GLASSWARES AND PLASTICWARES:

1. Routine washing: dilute bleach followed by drying in an oven, soaking in 20% Nitric Acid solution for 12- 24 hours
and soak in Acid dichromate solution.
2. For blood clots: soak in 10% NaOH
3. For new pipets: soak in 5% HCl or 5% HNO3
4. For metal ion determination, soak in 20% HNO3
5. For grease, soak in any organic solvent or 50% KOH
6. For permanganate stains, soak in 50% HCl or a mixture of 1% Fe2SO4 in 25% H2SO4
7. For bacteriologic glassware: soak in 2-4% cresol solution followed by autoclaving and thorough washing.
8. For iron determination, soak in 1:2 dilution of Conc. HCl solution or 1:3 dilution of Conc. HNO3.

GENERAL WASHING PROCEDURES:

1. Soak glassware in soapy water or dilute bleach detergent Rinse with tap H2O 3X Rinse
with dist. H2O oven dry @ > 140OC
2. Soak glassware in acid dichromate overnight rinse with dilute ammnonia rewash
according to the procedure.
Acid Dichromate preparation: Dissolve 50g sodium dichromate in 50 mL dist. H2O; Add to 500 mL conc.
H2SO4
3. Soak glassware in 20% HNO3 for 12 – 24 hrs rewash according to the first procedure.

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MEASURING DEVICES:

PIPETS:
✓ they are usually used to transfer volumes of 20 mL or less
CLASSIFICATION ACCORDING TO GRADUATIONS:

A. TRANSFER PIPETS: is designed to transfer a KNOWN volume of liquid.

1. Volumetric or Transfer pipet

✓ used to transfer aqueous solutions & non-viscous samples
✓ self-draining
✓ has the greatest degree of accuracy and precision
✓ read at the lower meniscus
✓ with bulb at the center
✓ Should be used when diluting standards, calibrators, or QC material.

2. Ostwald-Folin pipet
✓ used for biologic fluids having viscosity greater than water
✓ blowout pipets
✓ Indicated by two etched continuous rings at the top.
✓ read on the upper meniscus
✓ bulb is closer to the delivery tip

3. Pasteur pipets
✓ no calibration mark
✓ used to transfer solutions or biologic fluids without consideration of a specific volume
✓ Should not be used in any quantitative analytic technique.

4. Automatic (macro or micropipets)

✓ Is the most routinely used pipet in today’s Clinical Chemistry Laboratory.
✓ Automated/self-automated
✓ Advantages: safe to use, stable, ease of use, increased precision,
✓ time-saveing, less cleaning required.

3 GENERAL TYPES:

a. Air-displacement: relies on a piston for suction creation to draw the sample into a
disposable tip
b. Positive displacement: operates by moving the piston in the tip or barrel ; Sample enters directly upon
contact without air interference; NO need to replace delivery tip;
c. Dilutor/Dispenser pipets: obtain the liquid from a common reservoir and dispense it repeatedly; combines
sampling & dispensing functions

Dilutor/ Dispenser pipet

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B. GRADUATED OR MEASURING PIPETS – capable of dispensing several used to deliver a pre- determined volume
of liquid.

1. Mohr pipet
✓ calibrated between two marks; deliver between their calibration marks
✓ Does not have graduations to the tip.
✓ Tip should NOT touch the receiving vessel while the pipet is draining
✓ self-draining; with smaller orifice

2. Serological pipet graduated down to the tip

✓ blow-out pipet; with larger orifice
✓ Has an etched-ring ( or pair of rings) near the bulb end of the pipet

PIPET CLASSIFICATION ACCORDING TO DRAINAGE CHARACTERISTICS:

1. BLOW-OUT
✓ Has a continuous etched ring or two small, close, continuous rings located near the top of the pipet.

2. SELF-DRAINING
✓ No markings, the pipet is drained by gravity.

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CLASSIFICATION ACCORDING TO USE:

a. “To-Contain” (TC)
✓ Holds the particular volume but does not dispense the exact volume
✓ Requires rinsing
✓ calibrated with mercury
✓ usually a micropipette - Volumes are expressed in microliter
✓ Example: Sahli pipet

b. “To-Deliver” (TD)
✓ Delivers the exact volume indicated; Calibrated for the volume delivered
✓ fluid is allowed to flow freely with the pipet tip touching the inner wall of receiving vessel
✓ Designed to be drained by gravity
✓ Example: Mohr, Serologic, Volumetric Transfer pipets

CENTRIFUGE:

Centrifugation: process in which centrifugal force is used to separate solid matter from a liquid suspension; also
separate two liquid phases of different densities

Note: The speed of the centrifuge is related to the RCF by :

 RCF in grams= 1.118 x 10-5 x r x (rpm)2 ; or use nomogram

1.118 x 10-5= constant( determined from the angular velocity);

r = in cm ( measured from the center of the centrifuge axis to the bottom of the test tube shield or
bucket)

 RPM = tachometer or strobe light

TYPES OF CENTRIFUGE:

1. Horizontal-head or swinging-bucket centrifuge

✓ tubes placed in the cups of the rotor assume a horizontal plane when the rotor is in motion and vertical position
when at rest
✓ The rotor looks like CROSS with bucket
✓ The surface of the sediment is flat

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2. Angle-head or fixed angle centrifuge
✓ Tubes are held at a fixed angle from 25-40⁰ to the vertical axis of rotation
✓ Particles are driven outside and bottom of the tube and the surface of the sediment packs against the side and
bottom of the tube and the surface of the sediment is parallel to the shaft of the centrifuge

3. Ultracentrifuge
✓ High-speed centrifuge; its rotor can spin as high as 1000000 x g ; with mainly fixed angle rotors
✓ For the separation of lipoproteins
✓ requires a refrigerated chamber

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 Assessment No. 1:

1. What is used to measure the speed of a centrifuge?

2. What glassware is heatable to 900⁰C and withstand downshock from

900⁰C to ice water.

3. The type of water desired for use in test methods requiring

maximum accuracy and precision.

4. It is a chemical that has the highest purity and can be

measured directly to produce a substance of exact known concentration.

5. What centrifuge is used for the separation of lipoproteins?

6. What liquid is used for the calibration of TC pipettes?

7. This is a glassware with poor heat resistance but has a high

resistance to alkali.
8. It is the oldest method of water purification.

9. A property of glass that is dependent on boron oxide, nickel, and

ferric ion content.
10. A preparation of reagent grade water that uses an anion or cation
exchange resin.
11. These chemicals meet specifications set by the American
Chemical Society.

12. What type of water is used in chemistry, hematology, and

immunological testing?

13. What plasticwares are chemically resistant to most

substances except for aldehydes, amines, ethers, and hydrocarbons?
14. What is used for chemical sterilization of high quality
plastics?

15. What micropipet relies on a piston for suction to draw the

sample into a disposable tip?

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 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Activity No. 1
INSTRUMENTS USED IN CLINICAL CHEMISTRY 1

MATERIALS:

A. Equipment: B. Glasswares:

1. Analytical Balance 1. Volumetric Flask

2. Clinical centrifuge 2. Centrifuge tube
3. Hot air sterilizer 3. Folin-Wu tube
4. Interval timer 4. NPN tube
5. Pipet washer 5. BUN tube
6. Water bath 6. Cuvet / Stat fax tube

C. Pipets: D. Venipuncture Set:

1. Serological 1. Syringe, hypodermic needle, tourniquet

2. Ostwald-Folin 2. Vacutainer set (adapter, two-way needle, tube)
3. T.C. pipet
4. Volumetric pipet
5. Mohr pipet

PROCEDURE:
Examine the above- listed instruments and describe the use(s) of each in clinical chemistry.
DRAW AND LABEL:
Illustrate the above-listed Glasswares, Pipets and the Venipuncture Set. Draw and label the parts of all the
equipment listed.
GUIDE QUESTIONS:

Describe the above-listed equipment & instruments. Indicate the use of each in terms of Clinical Chemistry application.

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 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Activity No. 2
PIPETTING AND CALIBRATION OF PIPETS

MATERIALS:

Serological pipets, test tubes, beaker with water, test tube rack, vial with cover and filter paper or gauze

PROCEDURE:

A. Pipetting Technic
1. Select your pipet according to the nearest volume to be dispensed.
2. Place the pipet way down into the beaker containing water with the pipet calibration facing you.
3. Using a pipettol, suck the water beyond the 0 mark. Avoid introduction or formation of bubbles. Note:
Never pipet with your mouth
4. Remove the pipettol and immediately place your index finger on top of the pipet applyling enough
pressure to prevent the sample from flowing down.
Note: Do not use your thumb.
5. With the other hand, wipe the sides of the pipet with filter paper or tissue paper to remove the excess
water clinging at the sides of the pipet.
6. Adjust the contents of the pipet to the 0 mark by gently releasing the index finger.
Note: Reading should should be made at the - lower meniscus for non-viscous solution and
- upper meniscus for viscous solution.
7. Dispense the desired volume of water into the test tube. Note:
Always maintain a vertical position while dispensing.
8. Repeat above procedure using different pipets and dispensing different volumes of water until you have
mastered the use of the pipet.

B. Calibration of Pipet
Calibrating agents: a. water – for TD (To Deliver) pipets
b. Mercury – for TC (To Contain) pipets

1. Weigh a vial with its cover.

2. From the flask of distilled water containing a thermometer, measure a sample of water.
3. Transfer the sample into vial and again obtain its weight. Use the data to determine the actual capacity
of the pipet.
Actual Capacity = wt. of vial w/ water – wt. of empty vial

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 4. Table:

Temperature Weight of 1 ml. water Weight of 1 ml. Mercury(Hg)

20 0.9972 13.547
21 0.9970 13.545
22 0.9968 13.543
23 0.9966 13.541
24 0.9964 13.539
25 0.9961 13.537
26 0.9959 13.534
27 0.9956 13.532
28 0.9954 13.530
29 0.9951 13.528
30 0.9948 13.526

5. Multiply the weight (from the table) by the volume of the pipet being checked to get the
Theoretical weight.
6. Compute for the percent of error as follows:
% error = Actual capacity – Theoretical weight X 100%
Actual capacity
7. Establish your upper and lower limits.
8. Conclude whether the pipet used is properly calibrated (if the actual capacity is within the limits) or not
properly calibrated (if outside the limits).

GUIDE QUESTIONS:
1. Why should mouth pipetting be avoided?
2. In relation to pipetting, what is a parallax error?
3. Differentiate gravimetric from spectrophotometric method of pipet calibration.
4. Why should pipets be drained in a vertical position?

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 CHAPTER II: LABORATORY SAFETY MANAGEMENT

SAFETY – freedom from harm or danger; absence of risk

CONCEPTS ON THE PSYCHOLOGY of SAFETY

1. Safety is the concern & responsibility of everyone (employees, administrators, staff)

2. Injuries & accidents affect the morale and threaten the emotional & physical well-being of all persons
involved as well as co-workers
3. Accidents are expensive
4. Causes of injuries or accidents:
• Negligence or carelessness
• Ignorance or lack of orientation
• Fatigue or stress
• Lack of awareness
• Haste / cramming
• Inexperience
• Loss of focus
5. Implementation of preventive measures are essential
6. Development of biological & chemical exposure plans

OSHA REQUIREMENTS (Occupational Safety and Health Administration): Public Law 91-596
✓ Goal: to provide all employees (clinical laboratory personnel included) with a safe work environment
❖ Healthcare institutions are vested with moral responsibilities to provide:
- Safe environment / workplace ( Safety showers, chemical fume hood)
- Training
- Protective equipment and gadgets (laboratory gown, gloves, shoes)

OSHA STANDARDS

YEAR STANDARD
1984 Respiratory Protection Standard
1987 Hazard Communication Standard : “Right – to – Know Law”

1991 Occupational Exposures to Hazardous Chemicals in Laboratories – “OSHA Laboratory

Standard”:

1991 Bloodborne Pathogens Standard

1992 Formaldehyde Standard

(revised)

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 ❖ Licensure - A mandatory process by which a state grants permission to an individual or
organization to engage in a given occupation or business
❖ Registration - A process by which qualified individuals are listed on an official roster
maintained by a government agency
❖ Credentialing / Certification - A voluntary process by which a NGO grants recognition to an
individual who has met certain educational requirements and demonstrated competency by
examination
❖ Accreditation - A voluntary process of external review in which a private agency grants public
recognition to an institution that meets certain standards

REGULATORY AGENCIES:

1. Department of Transportation
2. Environmental Protection Agency
3. Food and Drug Administration
4. Department of Health
5. Occupational Safety and Health Administration

AGENCIES THAT ISSUE GUIDELINES & STANDARDS

1. American Association of Blood Banks

2. Centers for Disease Control and Prevention
3. International Organization for Standardization
4. Clinical and Laboratory Standards Institute

HAZARDS in the WORKPLACE

HAZARD
✓ a substance, situation or condition that is capable of inflicting harm to human health, property or system
functioning
✓ Capable of producing serious injury or life-threatening diseases

STRUCTURAL REQUIREMENTS

1. Design and Lay-out

✓ Consider location, traffic pattern, entrance & exit routes, positioning of the different sections in the clinical
laboratory

2. Structural Safety Rules

a. Building materials : concrete
b. Storm damage control
c. Fire prevention
d. Fire-fighting systems
e. Entrance & exit routes
f. Storage of flammable reagents
g. Blockage of hallways & doors
h. Ventilation system

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 3. Workflow process
✓ Consider minimum exposure to hazardous materials
✓ Structural barriers between testing & non-testing materials
✓ Traffic pattern control
✓ Areas containing hazardous materials
✓ Delivery & storage of reagents (arrangement)
✓ Collection & processing of the specimen
✓ Delivery to the testing site

4. Ventilation plan especially for Histopathology & Microbiology Sections

5. Location / ease of use / availability of safety fixtures and decontamination stations

TYPES OF HAZARDS IN A CLINICAL LABORATORY

TYPE SOURCE POSSIBLE INJURY

Biologic Infectious agents Bacterial, fungal, viral or parasitic infections

Sharps Needles, lancets, broken glasswares Cuts, punctures, or blood-borne pathogen

exposure
Chemical Preservatives and reagents Exposure to toxic, carcinogenic, or caustic
agents
Radioactive Equipment and radioisotopes Radiation exposure
Electrical Ungrounded or wet equipment and Burns and shock
frayed cords
Fire / Explosive Bunsen burners, alcohol lamps, organic Burns and disability (dismemberment)
chemicals
Physical Wet floors, heavy boxes, loitering Falls, sprains or strains
patients or personnel

I. BIOHAZARD (Biologic Hazards)

✓ Exposure occurs from ingestion, inoculation, tactile contamination, or inhalation of infectious material from
patients or their body fluids/tissues, supplies or materials they have been in contact with, or contaminated
needles, or by aerosol dispersion.

Preventive measures:
1. Use of personal protective equipment (PPE)
2. Handwashing before and after handling patient; after contact with specimen
3. Isolation of highly infective or susceptible patients
4. Proper disposal of waste

UNIVERSAL PRECAUTION – instituted by CDC (1985)

1983 – General guidelines for isolation; precautions in hospitals 1985 –
Blood specimens are considered potentially infectious
1987 – Blood & certain body fluids of all patients should be treated potentially infectious
1989 – All patients are assumed to be possible carriers regardless of the patient’s condition or state

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 SPECIMEN w/c are POTENTIALLY SPECIMEN w/c are USUALLY NOT
INFECTIOUS INFECTIOUS (unless visibly bloody)
Blood Feces
Pus & purulent fluids Nasal secretions
Seminal fluid Sputum
Vaginal secretions Sweat
Cerebrospinal fluid Tears
Pleural fluid Urine
Peritoneal fluid Vomitous
Pericardial fluid
Amniotic fluid
Breast milk

II. SHARP HAZARD

✓ Needles, lancets, probes of machines
✓ Never re-cap a needle
✓ Use needle disposal container / needle incinerator

* The Needlestick Safety and Prevention Act of 2000 – revises the Blood-borne Pathogen Standard of 1992
✓ Regulates occupational exposure to bloodborne pathogens, including HIV, HBV, and HCV.

III. CHEMICAL HAZARD

➢ Flammable, corrosive, explosive, carcinogenic, toxic, reactive reagents
➢ All chemicals should have MSDS (Material Safety Data Sheet)
➢ The MSDS must be consulted prior to the use of a reagent

Safety Practices:

a. PPE – Lab. Coats, aprons, goggles

b. Fume hoods- to contain and expel noxious and hazardous fumes from chemical reagents.
c. Storage – classify according to characteristics
✓ Storage cabinets must be below eye level
✓ Labeled with toxicity, specific hazard concern; manufacturer’s name, address, date of preparation, hazard
warning
d. Presence of a chemical safety officer

Note: The Globally harmonized System of Classification and Labelling of hazardous Chemical ( GHS)
incorporates universal definitions and symbols to clearly communicate specific hazards in a concise label format.

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 HAZARD CATEGORIES OF CHEMICALS

HAZARD CATEGORY CHARACTERISTICS EXAMPLES

CORROSIVES - Cause visible destruction of human - conc. HAc, H2SO4, HCl; conc.
tissue or injury upon contact or NH4OH, KOH, NaOH
inhalation.
- Chemicals with pH<2 or >12.

REACTIVES - Can spontaneously explode or ignite, or Ethyl ether & isopropyl ether ;
evolve heat, flammable or explosive gas Perchloric acid
under certain conditions
IGNITABLES Flammables: have FP <100 F or <37.8 Acetone, alcohols, ether, xylene,
C benzene
Combustibles: FP (=/>100 C) Flashpoint:
the lowest TO that produces sufficient vapor
to form an ignitable
mixture at the surface of the liquid
MUTAGENS Induce genetic mutations; cause changes in Radioactive isotopes, benzene,
the DNA molecule benzidine, heavy metals
TERATOGENS Cause physical defects in the developing
embryo
CARCINOGENS Upon prolonged/repeated exposure, may
promote the development of cancer cells

CLASSES OF HAZARDOUS MATERIALS (established by UN)

Class 1 : Explosives- ignitors, flares, ammunition, fireowrks
Class 2 : Compressed Gas- natural gas, fire extinguishers, propane Class
3 : Flammable Liquids- paints, fuels, acetone, alcohol
Class 4 : Flammable Solids- sulphur, matches, metal powders, activated carbon Class 5 :
Oxidizer materials- hydrogen peroxide, nitrates ( sodium, lead, ammonium) Class 6 : Toxic
materials- medical wastes, clinical wastes, acids
Class 7 : Radioactive materials- yellowcake, depleted uranium, medical isotopes Class 8 :
Corrosive materials- batteries, acid solutions, flux
Class 9 : miscellaneous materials- asbestos, vehicles, engines, dry ice.

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 CHEMICAL HAZARD IDENTIFICATION SYSTEM
(National Fire Protection Agency)

IV. PHYSICAL HAZARD

✓ Wet floors, heavy boxes, human traffic
✓ Eliminate clatter; clean spillage ASAP

Page 20 of 120
V. FIRE HAZARD
✓ First signs of fire (smoke, burning smell) : investigate then sound fire alarm
✓ Evacuate immediately; unplug all electrical gadgets
✓ Use fire extinguishers

CLASSES OF FIRES

Image taken from: fireprevention.utexas.edu

TYPES AND APPLICATIONS OF FIRE EXTINGUISHERS

Page 21 of 120
Strategies:

✓ Keep all flammable objects in separate rooms / storage areas

✓ Use fire-resistant building material or use materials that can contain fire for two (2) hours
✓ Availability of explosion – proof equipment
✓ Use vacuumized chambers when working with flammable reagents

Guidelines in Handling Flammable Reagents:

• Area: 5,000 sq. ft. = maximum of 10 gallons of flammable reagents

✓ To store 60 gallons BUT in a safety cabinet; if NONE, only 25 gallons are to be stored in safety cans

➢ SAFETY CANS

LIMIT: 1 pint of Class A flammable reagents ( highly combustible: e.g.: ether) 1 quart
of Class B flammable reagents ( Acetone, ethanol, methanol) 1 gallon of
other reagents ( xylene)

TRAINING AND PRACTICE

- Orientation and in-service training

- Hands-on use of fire extinguishers, fire blankets, emergency eyewashes, fire isolation
techniques
- Comprehensive techniques in identifying the fire
- Fight the FIRE or abandon the FIRE

VI. ELECTRICAL HAZARD

2 MAJOR ELECTRICAL HAZARDS in the LABORATORY:

A. Electrical equipment
B. Electrical wiring
➢ Defective equipment – unplug & discontinue usage; inform supervisor
➢ Decontaminate / disinfect before repair

Page 22 of 120
SAFEGUARDS recommended by NFPA, adopted by CAP, JCAHO and other fire districts:

1. No extension cords or outlet adapters

2. Equipment should be checked for compliance to electrical standards before use
3. Laboratory preventive maintenance programs:
a. Once a year examination of voltage and grounding
b. Current leakage
c. Broken, worn or frayed ends on plugs and cords
d. Review power needs of the laboratory and electrical receptacles should be adequate
e. Circuit breakers conveniently located & labeled
f. Electrical equipment should not be used in areas of flammable materials
g. Electrical safety must form part of the orientation & educational program of the laboratory

VII. RADIOACTIVE HAZARD

➢ Storage of radioactive materials: caution signs; access by authorized employees only.
➢ Exposure limit: (maximum permissible dose equivalents) 5000 mrem/year whole body
➢ Nonionizing radiation: microwaves, infrared, UV ( Less harmful because because its radiation has less
energy than ionizing radiation)

Clinical Chemistry by Bishop, 7th Ed.

VIII: MECHANICAL HAZARD PRECAUTIONS:

✓ Centrifuges must be balanced to distribute the load equally, never open the lid until the rotor stops
completely.
✓ Glass beads/ boiling chips should be added to help eliminate bumping/ boilover when liquids are heated
✓ Tongs or insulated gloves should be used to remove hot glasswares from ovens, hot bath, or water baths.

Page 23 of 120
 WASTE MANAGEMENT

⦿ Four basic waste-disposal techniques :

➢ flushing down the drain to the sewer system
➢ incineration
➢ landfill burial
➢ recycling

TYPES OF WASTES:

HAZARDOUS WASTES - may pose a threat to human health or the environment when improperly handled

INFECTIOUS WASTES - Anything that may harbor or transmit pathogenic organisms from individuals who may have a
communicable disease

MEDICAL WASTES - any solid, semisolid or liquid waste generated in diagnosis, treatment or immunization of
humans or animals in research or production or testing of biological samples
✓ Methods for treatment: incineration, steam sterilization, burial, thermal inactivation, chemical
disinfection, or encapsulation in a solid matrix.

CHEMICAL WASTES-
✓ Flush water-soluble substances down the drain with large quantities of water..
✓ Strong acids and bases should be neutralized before disposal.
✓ Flammable solvents: must be collected in approved containers and segregated into compatible classes.
✓ Solvents: Xylene and acetone: may be filtered or redistilled for reuse.
✓ Flammable materials: can be burned in specially designed incinerators
✓ Solid chemical wastes: bury in approved, permitted landfill.

WASTE CONTAINERS
COLOR CODE TYPE OF WASTE
Black Non-infectious DRY waste
Green Non-infectious WET waste
Yellow Infectious Pathological waste
Yellow with black band Chemical waste (heavy metals)
Red Sharps & pressurized containers
Orange Radioactive wastes

Page 24 of 120
 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Activity No. 3
LABORATORY SAFETY MANAGEMENT: AN OVERVIEW

OBJECTIVE: At the end of the activity, the students are expected to:

1. Recognize the different hazards in the clinical laboratory;

2. Appreciate the significance of laboratory waste management and the maintenance of a safe laboratory
environment;
3. Adhere to policies relating to laboratory in accordance to OSHA standards;
4. Demonstrate laboratory safety practices and proper waste disposal;

LABORATORY SAFETY MANAGEMENT:

Because of the nature of work performed, clinical laboratory personnel are exposed to a variety of hazards which
includes toxic vapors, flammable liquids, electric shock, corrosive substances, mechanical & ergonomic trauma and
risks which are associated with handling biological samples. It is thereby imperative that laboratory professionals and
students must understand the risks associated with these hazards. Safety begins with the recognition of hazards and is
achieved through the application of common sense, a safety- focused attitude, good personal behavior, good
housekeeping in all laboratory work and storage areas, and above all, the continual practice of good laboratory
technique (Bishop, Fody & Schoeff, 2018)
GUIDE QUESTIONS:

1. Briefly describe the following OSHA Standards:

a. Formaldehyde Standard
b. Blood-borne Pathogen Standard
c. Hazard Communication Standard
d. Occupational Exposure to Hazardous Chemicals (OSHA Laboratory Standard)
2. In relation to Safety Awareness, enumerate the responsibilities of the Employer and Employees in the clinical
laboratory.

Page 25 of 120
 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Experiment No. 4 LABORATORY SAFETY MANAGEMENT

Hazard Classification of Reagents & Biosafety OBJECTIVE: At

the end of the activity, the students are expected to:

1. Classify reagents based on their hazardous nature;

2. Characterize reagents according to the hazard identification system (HIS) of National Fire Protection Agency
(NFPA);
3. Distinguish the different types of fire extinguishers according to content and use;
4. Completely describe the different levels of biosafety in the clinical laboratory;

LABORATORY ACTIVITY:

* The laboratory instructor shall provide a pre-laboratory discussion of the types of reagents according to their hazard
nature and the characterization of reagents based on the hazard identification system established by the NFPA.

INSTRUCTIONS: Use a separate bond paper for the activity.

1. Prepare a list of the reagents that are commonly used in the laboratory.
2. In tabular form, classify the reagents according to their hazard nature. (Corrosives, Reactives, Ignitables,
Carcinogens, Mutagens)
3. Among the reagents which you have listed, choose three (3) and characterize these reagents following the Hazard
Identification System of NFPA.

GUIDE QUESTIONS:
1. Illustrate the HIS symbol. Describe the different colored areas in the symbol.
2. In tabular form, differentiate the different fire extinguishers according to their:
a. Color code
b. Use (indicate the class of fire on which these are used)
c. Content / additive
3. Completely describe the biosafety levels in the laboratory setting.
4. In tabular form, differentiate the biosafety cabinets according to:
a. Face velocity
b. Airflow pattern
c. Applications

Page 26 of 120
 CHAPTER III: PHLEBOTOMY

Desired learning outcomes:

At the end of this chapter, you should be able to:

A. Identify the methods of performing venipuncture.

B. Prepare blood sample ( serum and plasma).
C. Observe proper specimen handling.
D. Identify the sources of variations in specimen collection and processing. MICROSAMPLING –
collection of minute sample of blood through capillary puncture SITES:

✓ INFANTS up to 1 year of age

- Plantar surface of the big toe
- Median or lateral side of the heel
- Depth of puncture: up to 2.4 mm

✓ ADULTS:
- Middle or ring finger
- Puncture must be slightly off-center, perpendicular to the fingerprint
- Margin of the earlobe
- Depth of puncture: up to 3.1 mm

MACROSAMPLING – collection of a greater volume of blood from the veins or arteries

ARTERIAL PUNCTURE:
- Collection of arterial blood for blood gas analysis or blood pH determination
- Common sites: radial artery, brachial artery, femoral artery

https://www.mometrix.com/academy/arterial-punctures/

Page 27 of 120
VENIPUNCTURE:
- Collection of venous blood
-Most frequent site: Antecubital vein of the forearm

SITES:

✓ Infants up to 18 months: External jugular vein

Superior longitudinal sinus Temporal
vein
✓ 18 months to 3 years: Popliteal vein
Femoral vein
Long saphenous vein Ankle
vein
✓ 3 years to adulthood: Veins on the antecubital fossa
Wrist vein
Veins on the dorsal of hands & feet

METHODS OF PERFORMING VENIPUNCTURE:

I. OPEN SYSTEM – use of syringes

- The hub of the needle is color-coded, corresponding to its gauge
- The lower the gauge, the bigger the bore of the needle

II. CLOSED SYSTEM – use of an evacuated system (evacuated tube, two-way needle and adapter)
- Evacuated tubes are equipped with a hemogard (color-coded in accordance to the additive present)
- Multiple sampling can be carried out

SAMPLE PREPARATION

I. SERUM – liquid portion of clotted blood

II. PLASMA – liquid portion of anti-coagulated blood

III. PROTEIN-FREE FILTRATE (PFF): preparation involves the removal of proteins from any biological specimen to
prevent direct colorimetric interference by the formation of zwitterions at isoelectric pH where proteins exhibit
maximum precipitation and minimum solubility

Page 28 of 120
METHODS of PFF PREPARATION:

A. Physical Methods:
1. Heat
2. Ultracentrifugation
B. Chemical Methods

(ACID Method)
1. Folin-Wu
▪ Specimen: whole blood; plasma/serum
▪ 2/3 N Sulfuric acid
▪ 10% sodium tungstate
2. Hayden’s method
▪ Specimen: serum
▪ N/12 Sulfuric acid
▪ 10% sodium tungstate
3. Van Slyke - makes use of a pre-mixed acid
4. TCA (5%)

(BASE Method)
1. Nelson-Somogyi : 0.3N Ba(OH)2 & 5% ZnSO4

CONTAMINATION of SPECIMEN
❖ residual detergent in tubes
❖ Plasticizers in IV tubing and tube stoppers
❖ Cork stoppers & glass tubes
❖ Lead analysis: use lead-free, acid-washed containers

REMINDERS:
❖ Serum or plasma should be separated from cells within 2 hours of collection (unless collected in a gel
separator tube).
Rate of glycolysis: At ref temperature – 2 mg/dL
At room temperature – 7 mg/dL
❖ Allow red top tubes to clot sufficiently (20-30 minutes) before centrifugation to avoid fibrin strands.
❖ Centrifuge 10 ± 5 minutes at 1000-1200 × g.
❖ Keep tubes capped during centrifugation
❖ Lipemic specimens can be ultracentrifuged

SPECIMEN HANDLING REQUIREMENTS

1. FASTING
• Patients should not eat or drink anything (NPO) except water
• Glucose determination: 6 – 8 hours fasting
• Lipid Profile: 10 – 12 hours fasting
2. TIMED
• Specimen to be collected at a specified time
• Label the tube with the time the specimen was drawn
• Tests for Cardiac profile
3. PEAK / Post-dose
• Specimen collected when the highest serum concentration of a drug is anticipated
• Drawn 30 minutes to 1 hour after administration of the drug
• Therapeutic Drug Monitoring
4. TROUGH / Pre-dose
• Specimen collected when the lowest serum concentration of a drug is expected, usually before the next
dose is administered

Page 29 of 120
 5. CHILLED
• Chilling the specimen to slow down the metabolic process that will continue after blood is drawn
• Placed in crushed ice & water for even cooling
• Blood ammonia determination, blood gas analysis & Coagulation studies

6. KEEP WARM
• Keep specimen at body temperature or close to 37°C (incubator)
• For cold agglutinins
7. PROTECT FROM LIGHT
• Specimen should be wrapped in aluminum foil or a light-inhibiting container should be used
• Bilirubin, Vit. B12, Carotene
8. CHAIN OF CUSTODY
• For legal or forensic proceedings, sample may be used as piece of evidence
• Documentation of specimen handling begins with patient identification and continues until testing is
complete
o It must include date, time and identification of handler, all to be done in the presence of a witness
• DNA Analysis, Drug testing, Alcohol levels

REASONS FOR SPECIMENT REJECTION

✓ Hemolysis
✓ Lipemic / Lactescent serum
✓ Clots in an anti-coagulated specimen
✓ Partially-filled tubes
✓ Improper transport conditions
✓ Discrepancies b/w requisition & specimen label
✓ Unlabeled or mislabeled specimen
✓ Contaminated specimen/Leaking container

SPECIMEN INTERFERENCES

1. HEMOLYSIS
o Destruction of RBCs result in a plasma or serum appearing pink to red
o Hemoglobin concentration: exceeds 200 mg/L
o In-vivo hemolysis
o In-vitro hemolysis
o Interferes with:
▪ Enzyme and electrolyte assays (K+, Zinc and Mg+2)
▪ Serum albumin (↑): Bromcresol Green method
▪ Serum protein (↑): Biuret method
▪ Serum bilirubin (↓): Diazo method

2. ANTICOAGULANTS and PRESERVATIVES

o Potassium oxalate – dilution of plasma owing to water transport from the cells
o Anticoagulants that chelate calcium will inhibit various plasma enzyme activities
o Oxalates or citrates inhibit amylase activity
o Oxalates inhibit Lactate dehydrogenase and Acid phosphatase
o EDTA, oxalates and citrates will cause a decrease in calcium concentration

Page 30 of 120
3. ICTERIC SERUM / JAUNDICED SERUM

o Bilirubin interferes with the ff:

▪ Albumin assay: HABA method
▪ Cholesterol assay: Ferric chloride reagents
▪ Glucose determination: ortho-toluidine method
▪ Total Protein determination: Biuret method

4. LACTESCENT SERUM / MILKY SERUM / TURBID SERUM

o Interferes with the ff:

▪ Albumin assay: Bromcresol Green method
▪ Calcium : Cresolphthalein method
▪ Inorganic phosphates: Ammonium molybdate
▪ CK, bilirubin and total protein levels decrease
▪ Inhibits the activity of amylase, uricase and urease

5. PARTIALLY-FILLED TUBES

o For proper ratio of blood to anti-coagulant to be achieved, all tubes should be filled until the vacuum
is exhausted
o Most important for Coagulation studies

6. SPECIMEN CONTAMINATION

o Improper application of antiseptic on the site prior to venipuncture, or traces of Povidone iodine on the site of
puncture
o Aseptic technique is very important for blood cultures.
o Iodine increases K+ and Uric acid

SOURCES OF VARIATIONS IN SPECIMEN COLLECTION & PROCESSING

1. EXERCISE
Transient Effects: immediate fall and then subsequent increase in the concentration of free fatty acids
Longer-lasting Effects: Increase in activities of muscle enzymes such as Creatine kinase, aldolase, AST, and
Lactate dehydrogenase
Long-term physical training:
- Changes the levels of sex hormones
- During a six-month training, there is an increase in the mean plasma testosterone
concentration by 21%, the androstenedione concentration by 25%

2. PROLONGED FASTING – An 8 – 12 hour fasting is a must (GNFH or General Normal Fasting Hours) to
ensure that laboratory measurements are compatible with reference values.

3. DIET- Increase in the plasma concentration of potassium and triglycerides

✓ After 48 hours fasting: serum bilirubin increases
✓ After 72 hours fasting: plasma glucose decreases in healthy women to 45 mg/dL;
✓ After 72 hours fasting: increase Triglyceride, glycerol, fatty acids in men

4. ETHANOL INGESTION- Increase in plasma concentration of lactate, urate and the metabolites of ethanol
namely acetaldehyde and acetate
5. TOBACCO SMOKING- Up to 80% increase in carboxyhemoglobin, high plasma catecholamines,
, increased WBCserum cortisol
6. POSTURE - Obtain specimen in a supine or upright sitting position

Page 31 of 120
 7. OTHER FACTORS - Incorrect application of tourniquet, fist exercise cause an increase in the
concentration of lactate, enzymes, proteins, protein-bound substances

DIURNAL VARIATION:

➢ Lower at night:
• ACTH, Renin, Aldosterone, Insulin
➢ Higher in the afternoon & evening:
• GH, ACP,PT

➢ Iron, cortisol, Thyroid Stimulating Hormone, Eosinophil Count: peaks early to late morning &
decreases during the day

SHOCK and TRAUMA:

➢ Increased cortisol, aldosterone, catecholamine, glucagon and insulin

➢ Stress affects adrenal hormone secretion. Anxiety resulting in hyperventilation prior to venipuncture
will lead to disturbances in acid-base balance, an increase in the serum lactate concentration, and non-
esterified fatty acids, and leucocyte count.

SKIN COLOR:
➢ Blacks have increased CK, aldolase, and LDH

MENSTRUAL CYCLE:
➢ Increased cholesterol, protein, albumin, fibrinogen, Mg, Cl-, Na+, and PO4

AGE
➢ Newborn: bilirubin rises after birth and peaks at about 5 days
➢ Infants: lower glucose levels
➢ Uric acid peaks in men in their 20s but do not peak in women until middle age
➢ Total cholesterol: increases by 2 mg/dL ( 0.05mmol/L)/ year until midlife
➢ Postmenopausal women: increase cholesterol is attributed to a decrease in estrogen level
➢ Elderly: secrete less T3, PTH, aldosterone, and cortisol.
➢ After age 50: descrease testosterone among men, increase in pituitary gonadotropins ( FSH) among
women.

GENDER
➢ After puberty: Men have higher ALP, AST, CK, Aldolase levels than women
➢ Women: lower Magnesium, Calcium, Albumin, Hb, Serum iron, ferritin

Page 32 of 120
 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

EXPERIMENT NO. 5
PHLEBOTOMY

INTRODUCTION:

Phlebotomy – the drawing of blood – is an integral part of medical laboratory practice. It is good to
remember that no laboratory procedure will be any better than the quality of the specimen that is being tested. Each
step in the process of phlebotomy affects the quality of the specimen and is thus important for preventing laboratory
error and patient injury.

OBJECTIVE: At the end of the activity, the students are expected to:

- Given a request slip, rationalize the steps to be undertaken in patient preparation and specimen collection,
processing and handling.
- Perform venous of blood collection from an assigned patient following proper technique;
- Correctly label blood samples collected from the patient;
- Enumerate the precautions to be considered in proper specimen collection;
- Explain correctly proper specimen collection, handling and transport according to tests requested;
- Consistently maintain quality control in patient preparation, specimen collection, sample processing & handling;
- Establish guidelines on acceptability of blood samples submitted to Clinical laboratory.

PROCEDURE:

1. OPEN SYSTEM (Syringe Technique)

A. Preparation of the patient:

a. Identify the patient by checking identification band against labels and requisition form. Always ask conscious
patients his or her full name and birth date. Verify identity of an unconscious patient from a nurse or relative. If
identity is unknown, give temporary identification. Do not draw blood or any specimen without properly
identifying the patient.

b. If fasting specimen is required, confirm that the fasting order has been followed.
c. Address the patient and inform him or her
d. What is to be done. Remind the patient to avoid as much tension as possible.
e. Position the patient properly, depending on whether the patient is sitting or prone, for easy,
comfortable access to the antecubital fossa.

B. Preparation of the Venipuncture Set:

a. Assemble the equipment and supplies, including collection tubes, tourniquet, and preparations for cleansing
the area.
b. Select needle to be used
c. Fit the hypodermic needle to the nozzle of the syringe, and remove the plastic cap.

Page 33 of 120
 d. Check the condition of the needle by drawing back and forth the plunger. If there is difficulty in moving
the plunger, this means that the needle is clogged and should be replaced.
e. After the set is checked, replace the cap to prevent contamination of the needle. See to it that the sample
containers are adequate & complete. Never start the venipuncture technique if supplies and materials to be
used are not prepared.

C. Venipuncture Proper (Open system)

a. Ask the patient to make a fist to make the veins more palpable.
b. Select the suitable vein for puncture among the veins on the antecubital fossa.
c. Clean the venipuncture site with 70% alcohol. Begin at the puncture site and clean outward in a circular
motion. Allow the area to dry. Do not touch the swabbed area with unsterile object.
d. Apply tourniquet several inches (3-4 inches) above the puncture site. Never leave the tourniquet in place
longer than 1 minute.
e. Anchor the vein firmly. Use either the thumb and the middle finger or thumb and the index finger.
f. Remove the protective cap of the needle. Aim the needle with its bevel side up in line with the long axis of the
vein.

Note: The graduations of the syringe should be side up.

Never approach the vein from the side.
g. Hold the syringe placing the index finger on the hub of the needle then pierce the skin with the needle at
approximately 15O angle to the arm.
Note: Insert the needle smoothly and fairly fast to minimize patient discomfort.
h. If blood has entered the syringe, slowly pull back the plunger to aspirate the blood.
Note: Avoid excessive or quick pulling to avoid hemolysis or collapse of the vein. i.Should the
blood start to enter the syringe and then cease, the needle has either slipped out of the vein or gone through it.
If it has slipped out, carefully reinsert it by pulling back the needle slowly and draw it forward into the vein.
j. When the desired volume of blood has been obtained, release the tourniquet and instruct the patient to open his
or her fist.
k. Place a dry cotton ball over the site of puncture. Withdraw the needle then apply pressure onto the site.
l. Replace the cap of the needle and remove the needle from the nozzle of the syringe.
m. Dispense the collected sample into a test tube. Allow the sample to flow down the inner wall of the tube.
n. For tubes containing an anticoagulant, mix the blood sample and the anticoagulant by gentle inversion
of the tube.
o. Check the condition of the patient whether he or she feels faint and that the bleeding is under control.
p. Properly dispose all contaminated materials in appropriate receptacles.

D. CLOSE SYSTEM (Evacuated system)

a. Follow instructions as in syringe technique.

b. Place the needle (multiple or single) in the nozzle of the blood collection tube holder. Screw firmly.
c. Enter the vein smoothly and fairly fast to avoid discomfort of the patient.
d. Always maintain 15O angle with the bevel of the needle side up.
e. Follow the geographic arrangement of the vein. Always insert the needle along the long axis and never
along the side.
f. As soon as the needle is in the vein, ease the tube down forward in the holder as far as it will go. At the same
time, hold the needle in place firmly.
g. When the tube has filled, remove it by grasping the end of the tube and pulling it gently.
h. Withdraw the needle with the test tube holder while the site is covered with a sterile cotton.
i. Apply pressure at the site of puncture as soon as the needle leaves the vein.

Page 34 of 120
DRAW AND LABEL

GUIDE QUESTIONS:
1. What are the immediate, delayed and long lasting complications of venipuncture? (make use of the reference
book by McPherson and Pincus)
2. Differentiate an evacuated tube with & without a serum separating material in terms of advantage and use.
3. In multiple sampling, why is there a need to follow an “order of draw”?
4. Enumerate the appropriate order of draw in multiple sampling with the use of plastic evacuated tubes.

Page 35 of 120
 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Experiment No. 6 SAMPLE

PREPARATION

OBJECTIVE: At the end of the activity, the students are expected to:

- Correctly label blood samples collected from the patient;

- Explain correctly proper specimen collection, handling and transport according to tests requested;
- Consistently maintain quality control in patient preparation, specimen collection, sample processing & handling;
- Establish guidelines on acceptability of blood samples submitted to Clinical laboratory.

MATERIALS:

Evacuated system (Evacuated tubes – plain & with anticoagulant, two-way needle, adapter) clinical centrifuge,
test tubes, applicator sticks

PROCEDURE:

A. Whole Blood Preparation:

1. Collect blood with an anticoagulant.
2. This represents a whole blood sample and includes both solid and liquid portions of the blood.
B. Plasma preparation:
1. Collect blood by venipuncture.
2. Dispense blood into a tube with anticoagulant.
3. Mix by inversion.
4. Centrifuge the tube with anticoagulant at 2500 rpm for 5 minutes.
5. Separate the liquid portion of the packed red cells using a pasteur pipette. Transfer the liquid portion
into a clean tube. Avoid disturbing the preparation so that the red blood cells will not be aspirated. The
liquid is known as the plasma.
C. Serum Preparation:
1. Collect blood by venipuncture.
2. Dispense blood into a tube without anticoagulant.
3. Let stand for 30 minutes at room temperature to allow the blood to clot. Check for complete clotting
by tilting the tube. If blood does not move, it has completely clotted.
4. Gently rim the side of the tube with an applicator stick. Draw the applicator stick along the sides of
the tube as to retract the blood.
5. Centrifuge the tube without anticoagulant for 5-10 minutes.
6. Separate the liquid portion from the packed rd cells using a Pasteur pipette. This liquid portion is
now known as the serum.

Page 36 of 120
DRAW AND LABEL:

Draw the steps in the preparation of the different samples.

GUIDE QUESTIONS:
1. In tabulated form, differentiate serum from plasma.
2. Enumerate the most common causes of in-vivo and in-vitro hemolysis.
3. Why is fasting generally necessary before a chemical analysis could be done on a blood sample collected
from a patient?
4. Why is there a need to separate the serum from whole blood immediately? What are the changes that might
occur if separation is not done immediately?
5. Explain why proteins are removed from blood sample before chemistry assays are performed.
6. Describe techniques which are currently employed to eliminate proteins from serum.

Page 37 of 120
 CHAPTER IV: QUALITY CONTROL IN CLINICAL CHEMISTRY

Desired learning outcomes:

At the end of this chapter, you should be able to:

A. Define Quality Control. Quality Assurance Program.

B. Identify the areas and sysytem concepts of Quality Control.
C. Identify the procedures in the Quality Assurance Program
D. Be familiarized with the factors involved in Quality Control
E. Identify the Elements and Statistical Concepts in Quality Control
F. Know how to construct a QC Chart.

QUALITY – a degree to which a set of inherent characteristics fulfills requirements (ISO 9001:2008) QUALITY
CONTROL - part of quality management focused on fulfilling quality requirements (ISO 9000:2000)

• System of assuring the quality of total laboratory performance

• Involves the periodic systematic surveillance of people, tools, methods and reagents

QUALITY ASSURANCE – part of quality management focused on providing confidence that the quality
requirements will be fulfilled (ISO 9000:2000)

QUALITY ASSURANCE PROGRAM- Set of activities or plan that aims to maintain the highest degree of
excellence for the diagnosis & treatment of disease and maintenance of health

AREAS of QUALITY CONTROL:

1. All patients, laboratory personnel, laboratory equipment, and laboratory tests are involved.
2. The laboratory’s relation to other hospital departments
3. Laboratory policies and procedures should be collected in a manual to be revised at least once a year or
when procedures are changed.

SYSTEM CONCEPTS of QUALITY CONTROL:

1. Quality Control Surveillance System (QCCS) – establishes norms that must be met
2. Q.C. Corrective System – established to offer education of why errors occur; provide a program to remedy
defects
3. Objective Q.C. Parameters – established to prove that corrective measures have produced favorable
results.

Page 38 of 120
PROCEDURES in QUALITY ASSURANCE PROGRAM

A. CONTROL of PRE-ANALYTICAL VARIABLES

FACTORS:
1. Patient identification
2. Proper preparation of patient
3. Specimen collection, separation & processing

B. CONTROL OF ANALYTICAL VARIABLES

MAIN FACTORS:
1. Choice of analytical methodology
2. Calibration procedures
3. Proper documentation of analytical variables
4. Proper labeling & use of reagents
5. Preventive maintenance of analytical instruments
6. Periodic calibration of pipetting devices
7. Periodic checking of °T of refrigerator & heating units
8. Periodic checking of procedure manuals
9. Monitoring of technical competence
10. Inventory of control materials
11. Control assurance that safety measures are operational

C. CONTROL of POST-ANALYTICAL VARIABLES

FACTORS:
1. Verification of calculations of the final results
2. Check test results for possible transcription errors
3. Results should be easy to read & interpret
4. Timeliness of reporting values to patient chart
5. Procedures for informing physicians about results that require immediate attention

Page 39 of 120
FACTORS Involved in QUALITY CONTROL

1. STANDARD
o A substance of known composition
o Its value is established by an analytical procedure different from that used in the clinical
laboratory
2. CONTROL MATERIAL
o A substance which resembles the unknown specimen (patient’s sample)
o Analyzed daily together with the unknown
o The values obtained from the assays are used for the computation of the mean and the SD

Types of Control Material:

a. Commercial Control Sera

b. Pooled Sera
- Excess non-hemolyzed sera without gross lipemia are collected daily in the laboratory and pooled
for storage in the refrigerator
- When 1 – 2 liters are collected, centrifuge to remove gross contamination
- Filter and divide into aliquots of 5 mL each
- Stopper and store at -20°C
- Thaw as needed and mix well before use

ELEMENTS of QUALITY CONTROL:

1. ACCURACY – extent to w/c the measurement approximates the true value of the quantity being measured
2. PRECISION – a.k.a reproducibility; degree to w/c repeated results agree to each other
3. SPECIFICITY – ability of a method to detect a particular substance w/o the interference of some other
substances present in the sample
4. SENSITIVITY – ability of a method to detect even the smallest amount of that particular substance tested for
5. RELIABILITY – ability of a method to maintain its accuracy & precision over an extended period of time
6. PRACTICABILITY – degree to w/c the method is easily repeated

https://community.wanikani.com/t/precision-vs-accuracy/49996

Page 40 of 120
 STATISTICAL CONCEPTS in QUALITY CONTROL

1. ARITHMETIC MEAN
2. STANDARD DEVIATION
➢ A statement of the extent of random variation in any series of measurement
➢ A measure of the distribution of values around the mean

3. VARIANCE (s2)
⦿ Square of the standard deviation
⦿ Used to detect significant differences between groups of data
⦿ Determine contributions of various factors to the total variation
4. COEFFICIENT OF VARIATION
⦿ Percentile expression of the mean
⦿ Measure of the relative magnitude of variability
◾ CV = s (100)
X

Page 41 of 120
 Everytime the control is run, the value is plotted on the Levey-Jennings Chart

QUALITY CONTROL Associated Activities:

1. Assay of control samples
2. Instrument maintenance
3. Statistical Data Analysis
4. Proficiency testing survey

ASPECTS which the laboratory should avoid or minimize:

1. Analytical Bias
❖ Reported values do not fall along the line of slope when graphed
❖ Reported lab results do not correspond to the correct value

Types of Analytical Bias:

a) Proportional bias
b) Constant bias
c) Combined bias

NOTE: Problem on Accuracy (Bias) = Systematic Error

2. Random Analytical Variability

- Refers to laboratory analyses that are subject to imprecision
Types:
A. Proportional variability
B. Constant variability

NOTE: Problem on Precision (CV) = Random Error

3. ERRORS – can be systematic or personnel-related

TYPES OF ERRORS:

A. RANDOM ERRORS
⦿ Sources cannot be completely controlled or identified
⦿ Increase the extent of variability of results
CAUSES:
1. Pipets & volumetric glassware w/ manufacturing variation; electronic & optical variations in
instruments (e.g. spectrophotometers)

Page 42 of 120
 2. Variations in the cuvet
3. Variations in timing & °T control
4. Variations in light, evaporation & °T on serum sample
5. Interferences from other substances in the sample

B. SYSTEMATIC ERRORS
⦿ Displace the mean value on one direction (may be up or down), but do NOT affect the overall variability
as shown by the SD value
CAUSES:
1. Aging phenomena – decomposition of reagents during storage
2. Personal bias of the analyst
3. Laboratory bias
4. Inter- & intra-individual bias
5. Experimental errors or changes in the methods

TYPES OF SYSTEMATIC ERRORS:

1. TREND - Series of values on the control chart that continue to increase or decrease for at least a period of
six (6) consecutive days

CAUSES of UPWARD TREND:

o Expired lamps/photocells
o Denatured standard
o Contamination of reagents

CAUSES of DOWNWARD TREND:

o Standards that are too concentrated
o Contamination of reagent

2. SHIFT - Values that distribute themselves on one side of the mean for at least six (6)
consecutive days

CAUSES of UPWARD SHIFT:

o Partial electrical failure in instrument
o New standard is over-diluted
o Denatured standard that has stabilized
o Improperly prepared reagent
o Inaccurate timer
o Deteriorated indicator
o Dirty glassware
o Overheating in °T-sensitive analysis

CAUSES of DOWNWARD SHIFT:

o Increased concentration of standard & reagents
o Contaminated reagents
o Inaccurate timer
o Under-heating in analysis
o Contaminated glassware

Page 43 of 120
 DIVISIONS of QUALITY CONTROL

⦿ INTRALABORATORY / INTERNAL
✓ Q.C. procedures performed on a daily basis within individual laboratories
✓ Monitoring is carried out using:

o Levey – Jennings Chart

o Westgard Multi-rule Chart
o CUSUM Technique

⦿ INTERLABORATORY / EXTERNAL
✓ Performed on a less frequent basis (e.g. 3x a year) to compare performance b/w or among
laboratories

QUALITY CONTROL TECHNIQUES

QUALITY CONTROL CHARTS – graphical representations that display the control observation as a function or time
1. LEVEY – JENNINGS CHART
⦿ Control results are plotted on the Y-axis (ordinate) vs time on the X-axis (abscissa)
Interpretation:
o IN-CONTROL:
◾ All control values are w/in ± 2SD
◾ One outlier in 20 determinations
o OUT-OF-CONTROL:
◾ Presence of two or more outliers
◾ Presence of a Trend
◾ Presence of a Shift

⦿ Outlier - a control value that goes beyond ±2SD

CAUSES:
o Contamination of a single specimen
o Faulty pipette
o Incorrect dilution of test

Page 44 of 120
2. WESTGARD MULTIRULE CHART
⦿ Uses a series of control rules for interpreting data
⦿ False rejection is kept low
⦿ Error detection is improved
⦿ Analyse samples of the control material by the analytical method to be evaluated (at least 20 days)
WESTGARD RULES:
12S One control observation exceeds ±2SD Warning
22S 2 consecutive values exceed ±2SD Systematic error
13S One value exceeds ±3SD Random error
R4S One value exceeds +2SD & the other value Random error
exceeds -2SD
41S 4 consecutive values exceed ±1SD Systematic error
10X 10 consecutive values fall on one side of the Systemic error
mean

SIX SIGMA- seeks to identify, measure, and eliminate the large gaps of inefficiency in a process
⦿ Hands-on process with the single mantra of “improvement”
⦿ Improved: performance
quality
bottom line
customer satisfaction
employee satisfaction
⦿ The # of defects ( errors) per million opportunities (DPMO) is measured
3. CUMULATIVE SUM (CUSUM) CHART
⦿ Provides a display of the differences b/w the observed values & the expected mean
⦿ Used to monitor small shifts in the process mean.
⦿ Obtain the CUSUM by adding the difference (value from the mean) to the CUSUM of the previous differences

Interpretation:
o Examine the slope of the CUSUM line
o A steep slope suggests the presence of SE (Out of Control)
o DECISION LIMIT CUSUM – found in clinical Laboratories with microcomputer Q.C. programs

https://www.qimacros.com/control-chart/cusum-chart/

Page 45 of 120
4. YOUDEN PLOT / SCATTER DIAGRAM
⦿ Laboratory’s observed mean for material A (Y-axis) compared to the observed mean for material B (X-axis)
⦿ Information about the nature of the SE can be obtained when two diff. control materials have been analyzed

https://www.itl.nist.gov/div898/handbook/eda/section3/youdplot.htm

OTHER QUALITY CONTROL TOOLS

1. CHECK SHEET
- Presents information in an efficient, graphical format
- Accomplished with a simple listing of items

2. PARETO CHART
- Used to identify factors that have the greatest cumulative effect on the system

3. CAUSE & EFFECT DIAGRAM

- Ishikawa or Fish bone diagram - Used to associate multiple possible causes with a single
effect
- Primary branch
- Major branches
- Minor branches

4. FLOW CHART
- Pictorial representations of a process
- Used in identifying where errors are likely to be found in the system

5. HISTOGRAM
- Provides a simple, graphical view of accumulated data, including its dispersion & central tendency
- Ease of construction

Page 46 of 120
 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Experiment No. 7
CONSTRUCTION OF A REFERENCE CALIBRATION CURVE

OBJECTIVE: At the end of the activity, the students are expected to know how to prepare a reference calibration curve
and appreciate its use in clinical chemistry work.

PROCEDURE:

1. A series of standards of varying concentrations are prepared.

2. Each standard is treated in the same manner as patient’s sample, and is run through the
procedure.
3. The instrument reading (O.D. or %T) or other end point for each standard is plotted against its known
concentration on graph paper.
4. The concentration of the standard reagents are placed in a horizontal line and the instrument reading
on the vertical line.
Note: Use linear graph paper for the OD, and semilog graph paper for %T
5. The plotted points are then connected together by line. Note:
The “curve” should be linear.
6. To find the concentration of an unknown sample, locate the instrument reading on the vertical line and
then extend this reading till it intersects the slanting line.
7. From the point of intersection, extend the line downwards the horizontal line containing the
concentration values of the standards.
8. Take the reading. This indicates the concentration of the unknown sample. Note:
Set up a new standard curve with each new lot of reagent.

GUIDE QUESTIONS:
1. Describe the following:
a. Primary Standard Reagent
b. Secondary Standard Reagent
c. Standard Reference Material
2. Describe the importance of including a standard whenever blood chemistry analysis (analytical run) is performed
3. What is the use of a reference calibration curve?

Page 47 of 120
 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Experiment No. 8 CONSTRUCTION

OF A QUALITY CONTROL CHART
(Levey-Jennings Chart) OBJECTIVE:

At the end of the activity, the students are expected to:

1. Construct a Levey-Jennings Q.C. Chart following established guidelines

2. Interpret whether the analytical method being assessed is within or out-of-control based on the prepared
Q.C. Chart
3. Describe other Quality Control Tools which are used in Clinical Chemistry & other fields in
laboratory medicine

MATERIALS:Linear graph paper

PROCEDURE:

1. The results of at least 20 determinations are recorded (X)

2. Determine the arithmetic mean ( X ) of the values by taking the sum of the values divided by the number of
results.
3. Subtract each result in turn from the mean value ( X – X )
4. Square the differences so obtained ( X – X )2
5. Find the sum of the squared differences and divide by one less than the original number of results.
6. Extract the square root of the value obtained in no. 5. This represents the standard deviation

S.D. = (X - X)2
n–1

7. Determine the control limits (±1SD, ±2SD & ±3SD).

8. On the graph paper, a horizontal line is drawn to identify the mean value. The control limits are likewise
established. The control values are plotted on the Y-axis (ordinate) versus time/day on the X- axis (abscissa).
9. Use lines to connect the control values.
10. Interpret whether the analytical method is within or out of cont rol
based on the distribution of the control values on the chart.

STATISTICAL TOOLS:

1. Arithmetic mean
The measure of the center of distribution. It is the sum of the Values divided by the number of
values. The mean of a sample of values is calculated using the formula:

X = X
n
Where: X = mean
X = the sum of the observed values, from the first through the last observation
n = total number of observations

Page 48 of 120
2. Variance
It is desirable to have information regarding the spread or scatter of values about the center. Perhaps the
most frequently used measure of dispersion is the variance. When reference is made to a population, the
variance is denoted by the use of s2. The variance of a sample is denoted by s2 and is defined as a sum of
the square of the deviation of the observation from the mean divided by the total number of observations
less 1.

The greater the scatter of numbers from the center of distribution, the greater the magnitude of deviation
and the larger the variance. The formula for the variance of a sample is:
S2 = (X – X)2
n- 1

Where: s2 = variance of a sample of values

(X-X)2 = the square of the deviations of the observations from
the mean of the observations summed over the number of observations n
= total number of observations
3. Standard deviation
Standard deviation is the measure of the dispersion of values around the mean.

S.D. = (X - X)2
n–1

4. Coefficient of Variation
The standard deviation expressed as percentage of the mean. Frequently, it is to compare relative
variability between different sets of data. The data may result from the use of different series f specimen,
different methods, or different analysts. Comparison of the different data sets cannot be made by directly
comparing their means and standard deviation because the magnitude of the statistics and units in which
the results are expressed may vary from one set of data to another. Such comparison can be made,
however, if the standard deviation of each set is expressed as a percentage of the mean. The statistic
appropriate for such comparison is the coefficient of variation. The coefficient of variation provides a
measure of relative variability and is calculated as:

C.V. = s (100)
X

Where: C.V. = coefficient of variation

S = standard deviation
X = mean of the sample

5. Median
Another frequently used measure of central tendency is the median. If the observed values are arranged in
increasing order, the median is the middle observation. In other words, the median is the value of the
measured variables below which half the observation fall. If the number of obtained values is odd, there will
be a unique median. The value can be found by taking the (n+1)/2 value from either end in the ordered
sequence. When the number of values is even and there is no middle observation, the median is defined as
a arithmetic mean of the 2 middle observations.

Page 49 of 120
 6. Mode
The mode is the value that occurs most frequently in a list of items of data. It is not affected by extreme
values or outliers.

INSTRUCTIONS: Given the following data, prepare a Levey-Jennings Q.C. Chart. On a separate bond paper, provide
the solutions for the computation of the mean, S.D. and the control limits. Use a graph paper for the preparation of the
chart. Provide an interpretation of the analytical run.

CONTROL VALUE (X) (X - X) (X - X)2

GUIDE QUESTIONS:
1. Describe the following characteristics of an analytical method:
a. Practicality
b. Reliability characteristic
c. Analytical range
d. Analytical sensitivity
e. Detection limit
f. Blank readings
g. Analytical specificity
h. Interference
i. Recovery
j. Precision
2. What is a quality control material? Indicate its use in clinical chemistry work.
3. Distinguish between a commercially-prepared control material from in-house quality control material in terms of a)
method of preparation, b) advantages and c) disadvantages.

Page 50 of 120
 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Experiment No. 9 CONSTRUCTION

OF A QUALITY CONTROL CHART
(Westgard Multi-rule Chart)

OBJECTIVE: At the end of the activity, the students are expected to:

1. Construct a Westgard Multi-rule Q.C. Chart following established guidelines;

2. Interpret whether the analytical method being assessed is within or out-of-control based on the
established control rules;
3. Completely describe the Six Sigma and the Lean process for Quality Control.

PROCEDURE:

DAY CONTROL VALUE (X – X) (X – X)2

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

GUIDE QUESTIONS:
1. Enumerate the control rules for the Westgard Multi-rule quality control chart.
2. Describe the Six Sigma process as a quality control tool.
3. Describe the Lean process as a quality control tool.

Page 51 of 120
 CHAPTER V: ANALYTICAL METHODS & INSTRUMENTATION

Desired learning outcomes:

At the end of this chapter, you should be able to:

A. Discuss the principles of the different analytical methods in Clinical Chemistry 1.

B. Identify the components and functions of each instrument used in Clinical Chemistry.
C. Identify the factors affecting each analytical method.
D. Discuss the methods and principles of Electrophoresis.
E. Discuss the principles and types of electrophoresis,electrochemistry, isoelectric focusing, and
automation.

❖ Based upon measurements of radiant energy emitted, transmitted, absorbed, or reflected under controlled
conditions.

TERMINOLOGIES: ELECTROMAGNETIC

ENERGY
✓ radiant energy; photons of energy travelling in a wavelike manner
✓ The shorter the wavelength, the higher the electromagnetic energy.

Types of Electromagnetic energies:

1. Cosmic rays
2. Gamma rays
3. X-rays
4. Ultraviolet ( UV)
5. Visible light
6. Infrared (IR)
7. Radio, TV, microwave, etc.

Page 52 of 120
WAVELENGTH
✓ distance between two peaks as the light travels in a wavelike manner
✓ Expressed in nanometers (nm), angstroms (Å), and millimicron (mµ)
✓ 1 nm = 10 Å ;1 nm = 1 mµ

Kinds of Wavelength:

1. Visible spectra = 340 nm – 700 nm

2. Non-visible spectra = below 340 nm (ultraviolet region)
= above 700 nm (infrared region)

COLORIMETRY
✓ Measurement of the wavelength and the intensity of electromagnetic radiation in the visible region of the spectrum.
✓ Used for identification and determination of concentrations of substances that absorb light.

Kinds of Colorimetry:

1. Visual Colorimetry – relies on visual acuity to determine end-point

2. Photoelectric Colorimetry

COLORS & COMPLEMENTARY COLOR OF THE

ULTRAVIOLET & VISIBLE REGION

WAVELENGTH REGION NAME COLOR ABSORBED COMPLEMENTARY

COLOR (COLOR OF
SOLUTION)
180 - 220 Short UV Not visible -
220 - 340 Short UV Not visible -
340 - 430 Visible Violet Yellow green
430 - 475 Visible Blue Yellow
475 - 495 Visible Green blue Orange
495 - 505 Visible Blue green Red
505 - 555 Visible Green Purple
555 - 575 Visible Yellow green Violet
575 - 600 Visible Yellow Blue
600 - 620 Visible Orange Green blue
620 - 700 Visible Red Blue green

Page 53 of 120
 SPECTROPHOTOMETRY and FILTER PHOTOMETRY

a. SPECTROPHOTOMETRY
✓ measurement of light intensity in a much narrower wavelength
✓ Makes use of prisms and/or diffraction gratings as monochromator to disperse the radiant energy into a continuous
spectrum & isolate radiant energy of desired wavelength

b. FILTER PHOTOMETRY – measurements of light intensity of multiple wavelength

✓ It makes use of filters (interference or transmission) to isolate part of the spectrum

A. PRINCIPLE of SPECTROPHOTOMETRY (LAW of COLORIMETRY)

Light passes through a monochromator to provide a selection of the desired region of the spectrum to be used
for measurements. Slits are used to isolate a narrow beam of light and to improve its chromatic purity. The light next
passes through an absorption cell where a portion of the radiant energy is absorbed, depending upon the nature and
concentration of the solution. Any light not absorbed is transmitted to a detector, which converts the light energy to
electrical energy that can be registered on a meter or a digital read-out.
1. BEER’S LAW: The concentration of the solution is directly proportional to the amount of light absorbed
and inversely proportional to the logarithm of transmitted light

%T = ratio of the radiant energy transmitted, divided by the radiant energy incident on the sample Absorbance
/ Optical Density (O.D.) = the amount of light absorbed

2. BOUGUER’S LAW or LAMBERT’S LAW: Absorbance is directly proportional to the length of light path

A = abc

BEER- LAMBERT LAW- the concentration of a solution for the known path length is directly proportional to its
absorption of light.

https://sciencenotes.org/beers-law-equation-and-example/

Where: A = absorbance
a = proportionality constant or molar absorptivity or extinction coefficient. Constant for a given compound at a given
wavelength under prescribed condition of solvent, temperature, pH, etc.
b = length of light path in cm
c = molar concentration of absorbing substance

Page 54 of 120
 Internal Parts of a SINGLE BEAM- SPECTROPHOTOMETER

Image taken from: simulab.ltt.com.au

A – Light Source E – Cuvet

B – Entrance slit F – Detector
C – Monochromator G – Meter
D – Exit slit

B. COMPONENTS:

1. LIGHT SOURCE – provides a continuous spectrum of white light which can be separated at different
wavelengths
Types:
A. Tungsten Iodide lamp – produces energy wavelength from 340 – 700 nm (visible region); used for
moderately diluted solution
B. Quartz Halide lamp – contains small amounts of halogen such as iodine to prevent the
decomposition of the vaporized tungsten from the very hot filament
C. Deuterium Discharge lamp – provides energy source with high output in the UV range (down to 165 nm)
D. Infrared Energy source – used above 800 nm
Examples: Merst glower – an electrically heated rod of rare earth element oxides Globar –
uses silicon carbide
E. Mercury Vapor lamp – emits narrow bands of energy at well-defined places in the spectrum (UV and
visible)
F. Hollow Cathode lamp – consists of a gas-tight chamber containing anode, a cylindrical cathode,
and inert gas such as helium or argon

2. ENTRANCE SLIT – isolates a narrow beam of radiant energy; prevents stray light from entering the
monochromator

3. MONOCHROMATOR – wavelength selector; isolates radiant energy of desired wavelength and excluding
that of other wavelengths

Monochromator bandpass: the range of wavelengths transmitted and is calculated as width at more than
half the maximum transmittance.

Page 55 of 120
 TYPES OF MONOCHROMATOR:

A. Prism
✓ wedge-shaped pieces of glass, quartz, NaCl, or some other material that allows transmission of light
✓ Disperses white light into a continuous spectrum of colors by refraction
✓ Produces a non-linear spectrum. The longer wavelengths are close to each other and those of shorter
wavelengths are widely spaced.
✓ Glass prisms are for visible region while quartz prisms are for the UV region

B. Diffraction Gratings
✓ consist of a thin layer of aluminum-copper alloy on the surface of a flat glass plate that has many small parallel
grooves ruled into the metal coating
✓ Rays of radiant energy bend (refract) around the sharp edges of the grooves
✓ Extent of refraction varies with the wavelength

C. Transmission Filters
✓ colored glass or colored gelatin sandwiched between two glass plates
✓ Light outside the transmission band are absorbed by the colored material
✓ Band pass is 35 – 50 nm or more

D. Interference Filters
✓ Produce monochromatic light based on the principle of constructive interference of waves.
✓ dielectric material (e.g. NaF) sandwiched between two half-silvered pieces of glass
✓ The thickness of the layer determines the wavelength of energy transmitted.
✓ Band pass is 10 – 20 nm

4. ANALYTICAL / ABSORPTION CELL / CUVETTE – used to hold the solution whose concentration is to be
measures

Types:
a. Borosilicate glass – for solutions that do not etch glass
b. Quartz or plastic – does not absorb UV radiation at wavelength below 320 nm
c. Alumina silica glass – good for 340 nm and above (visible region)

5. DETECTORS – measure light intensity by converting light signal into electrical signal

Types of Detectors:

a. Barrier-Layer cell (Photocell or Photovoltaic cell)

✓ Composed of a film of light sensitive material (e.g. Selenium) on an iron plate with a transparent layer of silver
✓ When light passing through the semi-conductive metal layer falls upon the Selenium surface, electrons are
released in proportion to the intensity of light and are collected to the silver layer to produce a negative charge

b. Photoemissive tube or Phototube

- Has photosensitive material that gives off electrons when light energy strikes it
- Consists of 2 electrodes (cathode and anode) sealed in an evacuated glass
c. Photoconductive tube or Photoresistive tube
- A device whose electrical resistance decreases as the level of incident light is raised
- Cadmium sulfide or cadmium selenide are the light-sensitive materials typically used for the visible
region
- Does not require an external power source
d. Photomultiplier tube
- Capable of significantly amplifying a current
- The cathode is a negative light-sensitive metal that absorbs light and emits electrons in
proportion to the radiant energy that strikes the surface
- Electrons go to the dynodes, where electrons produce 4 – 6 additional electrons
- The electrons are collected at a final electrode, the positive anode

Page 56 of 120
 Advantages: - rapid response time
- very sensitive
- low fatigue

6. READ-OUT DEVICES – electrical energy from a detector is displayed

a. Direct reading system – the output of the detector is used to drive a sensitive meter directly without
further amplification
b. Null Point System – the output of the detector is balanced against the output of a reference circuit
c. Digital Read-out – numerical display of absorbance or converted values of concentrations
d. Microprocessors

DOUBLE BEAM SPECTROPHOTOMETERS

1. Double Beam-In-Space
• All components are duplicated except the light source
• The beams of light pass through different components but at the same time

Henry’s Clinical Diagnosis and Management by Lingrud

2. Double Beam-In-Time
• Uses a light beam chopper (a rotating wheel) – with alternate silvered sections and cut out sections,
inserted after the exit slit.

Henry’s Clinical Diagnosis and management by McPherson&Phincus, 22nd Ed.

Page 57 of 120
FLAME PHOTOMETRY / FLAME EMISSION SPECTROPHOTOMETRY / FILTER PHOTOMETRY

A. PRINCIPLE
✓ It involves the measurement of emitted light when electrons in an atom become excited by heat energy produced
by the flame. When these electrons return to their ground state, they emit light characteristic of the ions present.
✓ It is used primarily to determine concentration of sodium, potassium or lithium since these alkali metals are easy to
excite.

Sodium = yellow Rubidium = red

Potassium = violet Magnesium = blue
Lithium = red

B. COMPONENTS of the FLAME PHOTOMETER

1. GASES – mixture of hydrogen and oxygen gas

- acetylene
- propane
- natural gas
2. BURNER ASSEMBLY
a. Aspirator – draws sample into the flame
b. Atomizer (Nebulizer ) – creates a fine spray of sample solution to be fed into the flame of the burner
c. Flame – provides heat energy for excitation

Types of Burner:

A. Total Consumption Burner – aspirate sample directly into the flame, the gases are passed at high
velocity over the end of the capillary suspended in the solution
B. Premix Burner – involves the gravitational feeding of solution through a restricting capillary into an area of
high velocity gas flow where small droplets are produced and passed into the flame
3. INTERFERENCE FILTERS as MONOCHROMATOR

Na filter - transmit yellow light (589 nm)

K filter - transmit violet light (767 nm)
Lithium - transmit red light (761 nm)

4. DETECTOR – uses photocell as detector

The Internal Standard in Flame Photometry: Uses Lithium or Cesium

ATOMIC ABSORPTION SPECTROPHOTOMETRY (AAS)

A. PRINCIPLE:In AAS, the element is not excited in the flame but merely dissociated from its chemical
bond and placed in an unexcited state. The atom, at a lower energy level, absorbs light. The light
source emits radiant energy to be absorbed by the element.
- Measures the amount of light absorbed by ground state atom
✓ Used to measure aluminum, calcium, copper, lead, lithium, magnesium, and zinc.

B. COMPONENTS

1. LIGHT SOURCE – hollow cathode lamp, which produces a wavelength of light specific for the kind of metal in
the cathode
2. MECHANICAL ROTATING CHOPPER – modulates light beam coming from the hollow cathode lamp
3. BURNER – uses flame to dissociate the chemical bonds and form free, unexcited atoms

Page 58 of 120
 Two types of Burner:
a. Total Consumption burner – flame is more concentrated and can be made hotter, thus lessening chemical
interferences.
b. Pre-mix burner – gases are mixed and the sample is atomized before entering the flame and the large
droplets go to waste and not in the flame. It has less noisy signals with longer pathlength and greater
absorption and sensitivity.
4. MONOCHROMATOR – selects the desired wavelength from a spectrum of wavelength which could either be a
prism or a diffraction grating.
5. DETECTOR – uses photomultiplier tubes to measure the intensity of the light signal.
6. METER or READ-OUT DEVICE

Clinical Chemistry by Bishop, 7th Ed.

FLUORESCENCE SPECTROPHOTOMETRY

A. PRINCIPLE:

FLUORESCENCE – energy emission that occurs when certain compounds absorb electromagnetic radiation,
become excited and then return to an energy level that is usually slightly higher than their original level.

B. COMPONENTS:

1. LIGHT SOURCE – hydrogen discharge lamp or xenon lamp

2. MONOCHROMATORS:
a. Primary filter / Excitation filter – isolates the ultraviolet light
b. Secondary filter / Emission filter – isolates secondary emission (filter, prism or diffraction grating)
3. PHOTOMULTIPLIER
4. READ-OUT DEVICE

Page 59 of 120
GRAVIMETRIC METHOD
✓ separation of a substance in a pure form and then determining its dry weight
Example: Total Lipid determination

VOLUMETRIC / TITRIMETRIC METHOD

✓ the unknown sample is made to react with a known solution (titrating agent) in the presence of an indicator
Example: Chloride determination (Schales & Schales)

TURBIDIMETRY
✓ measurement of the amount of light blocked by a particulate matter suspended in solution (180° to the incident
beam)
✓ Used to quantify protein concentration in urine and CSF, used to measure antibiotic sensitivities from cultures,
used to detect clot formation in the sample cuvets

Factors affecting turbidimetry:

o Size and number of particles

o The depth of the tube
o Cross-sectional area of each particle

NEPHELOMETRY
✓ detection of light energy scattered or reflected toward a detector that is not in the direct path of the
transmitted light (90° to the incident beam)

• The factors affecting turbidimetric measurements are the same factors affecting nephelometric
measurements
• It is more specific than turbidimetry

SCINTILLATION COUNTER
✓ it is used to measure the disintegration of a radioisotope per minute

Page 60 of 120
 Types of Radiation:

1. Alpha – positively charged particles; resemble the nucleus of helium atom with a mass of 4
o Have very little energy
2. Beta – resembles an electron with both negative (β-) and positive (β+) charges but essentially no mass
o Exists in two forms: soft and hard beta
3. Gamma – a form of electromagnetic energy with no mass, only energy
o Exists in two forms: soft and hard gamma

Types of Scintillation Counters:

1. Solid Scintillation Counter/ Crystal Scintillation

✓ measures gamma radiation using thallium activated Sodium iodide crystal as scintillator and PM tube as detector
with pre-amplifier circuit

2. Liquid Scintillation Counter

✓ measures beta radiation using liquid flour as scintillator
✓ Used to count radionuclides that emit beta particles

Application for Scintillation Counting:

RADIOIMMUNOASSAY – an immunologic procedure involving the use of radioisotope

Substances involved in RIA:

1. Unlabelled antigen (Ag) – substance being analyzed
2. Radiolabelled antigen (Ag) – acts as label
3. 3. Antibody – provide binding site for the two antigens

Types of RIA:
1. Solid RIA
2. Liquid RIA

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 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Experiment No. 10
SPECTROPHOTOMETRY

OBJECTIVE: At the end of the activity, the students are expected to:

1. Describe the components of a spectrophotometer

2. State the working principle of spectrophotometry & other analytical methods
3. Properly manipulate the spectrophotometer
4. Observe proper handling and care of the spectrophotometer

PROCEDURE:

A. Examine the photometer (stat fax) and identify its parts

B. Operation of the Spectrophotometer (Stat fax machine)
1. Plug the line cord into the line power receptacle.
2. Position the power on-off switch at ON. Allow 15 minutes to warm up.
3. For obtaining absorbance readings, press the ABS key then ENTER.
4. Using the number keys, select the filter appropriate for the wavelength required for the analysis
5. When “Read blank tube” is shown on the monitor, insert the stat fax tube that contains the blank (water
blank, patient’s blank or reagent blank).
6. After the blank tube, insert the stat fax tube that contains the standard reagent or calibrator. Record
the absorbance reading.
7. Remove the standard tube and replace it with a stat fax tube containing the sample material. Again,
record the absorbance reading of the sample material.
Note: Make sure that the outside of the stat fax tube is properly wiped with gauze before inserting it into
the cuvet well.
8. Compute the concentration of the analyte being measured based on Beer’s formula

DRAW AND LABEL:

a. External parts of spectrophotometer (stat fax machine)

b. Diagram of the components of spectrophotometry
c. Diagram of the components of flame emission spectrophotometry
d. Diagram of the components of atomic absorption spectrophotometry
e. Diagram of the components of fluorescent spectrophotometry
f. Ion Exchange Chromatography

Page 62 of 120
GUIDE QUESTIONS:
1. State the principle of:

a. Flame emission spectrophotometry

b. Atomic absorption spectrophotometry
c. Fluorescent spectrophotometry
d. Densitometry
e. Ion exchange chromatography
f. Gas-Liquid chromatography
g. Polarography
h. Amperometry
i. Coulometry
j. Conductometry
k. Osmometry
l. Turbidimetry
m. Nephelometry
n. Immunochemistry

2. Discuss the types of interferences in atomic absorption spectrophotometry.

3. Completely describe calibration of a spectrophotometer for performance evaluation taking into
consideration the following:

a. Wavelength accuracy
b. Absorbance
c. Linearity
d. Stray Light

Page 63 of 120
 ELECTROCHEMISTRY

POTENTIOMETRY
✓ measurement of differences in voltage at a constant current
✓ The unknown voltage introduced into the potentiometer circuit opposes a known reference voltage
✓ The voltage of the unknown is measured by comparison to determine the voltage required to exactly oppose
the flow of current in the test circuit
✓ The relationship between the measured voltage and the sought-for concentration is shown by the
Nernst Equation

POLAROGRAPHY
✓ measurement of differences in current at a constant voltage
✓ Used to measure trace metals, oxygen, Vitamin C, and amino acid concentrationThe relationship between
the differences in current and voltage is shown by the Ilkovic Equation

COULOMETRY
✓ the measurement of the amount of electricity (in coulombs) at a fixed potential
✓ A coulomb is equal to a current flow of 1 ampere per second
✓ Used to measure chloride ions in serum, plasma, CSF, and sweat samples.
✓ The number of coulombs consumed can be related directly to the concentration of the unknown
✓ The relationship is expressed by the Faraday’s Law

AMPEROMETRY
✓ measurement of the amount of current that flows when a constant voltage is applied to the
measuring electrode

CONDUCTOMETRY
✓ measurement of the current flow between two non-polarizable electrodes between which a known electrical
potential is established

SEPARATION OF SUBSTANCES

I. PRECIPITATION
• Separation is based on solubility
• The precipitate is studied by:
a. Turbidimetric method
b. Chemical reaction (after being dissolved)
c. Gravimetric method

II. ULTRAFILTRATION and DIALYSIS

ULTRAFILTRATION DIALYSIS
- separates dissolved materials - separation of dissolved molecules
- removes particulate matter
- movement through a semi-permeable - movement through a semi-permeable
membrane driven by a force or pulled membrane more freely
through a vacuum
Cellulose esters Cellophane (regenerated cellulose) in sheets
Cellulose acetate or tubing
Polyamide
Polyvinyl chloride
Sheets, disks or hair thin fibers, conical
- For desalting, fractionation of protein solutions - Employed in continuous flow
and the preparation of pff automated systems
- used to purify or concentrate samples

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III. CHROMATOGRAPHY
Requires two (2) phases: (1) Solid support – coated or uncoated
(2) Mobile phase – flowing gas or liquid

TWO GENERAL TYPES of CHROMATOGRAPHY:

A. Adsorption Chromatography – molecules separated are adsorbed at the surface of a solid support or
flow with the mobile phase
B. Partition Chromatography – solid support is coated with a film of water or non-volatile organic liquid
Examples: TLC, GLC
1. Paper Chromatography

a. Solid or immobile phase – paper is composed of cellulose; the matrix of cellulose is bound to water
b. Mobile phase ( carries the sample through a bed
✓ organic solvent
✓ involves partition between water and organic solvent
✓ if the molecules are more soluble in the flowing solvent, the faster it will move along the paper
✓ if the molecules are more soluble in water, they do not move very fast
• Rf = ratio of the distance of movement by a compound to the distance of the solvent front

Rf = a
B
Where: a = distance travelled by compound from origin to front of spot b =
distance travelled by solvent

2. Thin Layer Chromatography

o Uses a flat sheet of chromatographic material
o Advantages over paper chromatography
• Solid Support: water bound to
a. Silica or silicic acid
b. Alumina – aluminum oxide + aluminum hydroxide

• Mobile Phase: organic solvent

Qualitative analysis: based on colors and positions

Quantitative analysis: remove spots and extract

3. Ion-Exchange Chromatography
o Separation is based on electrical charge
o Anion exchangers – capture anions
o Cation exchangers – capture cations

• Stationary / Immobile / Solid support

a. Aluminum silicate
b. Polysaccharide
c. Synthetic resins – polystyrene beads
• Mobile Phase: water

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 4. Gel Filtration / Molecular Sieve / Gel Permeation / Size Exclusion / Molecular Exclusion
o Separation is based on differences in molecular size
• Stationary phase
a. Polyacrylamide (plastic)
b. Sephadex (cross-linked polysaccharide)
c. Porous beads
• Mobile phase: flowing water

5. Gas Chromatography
Two general types:
a. Gas – Liquid Chromatography (GLC) – based on partition
b. Gas – Solid Chromatography (GSL) – based on adsorption

Two Phases:

• Stationary Phase – diatomaceous earth (silica) coated with a non-volatile organic liquid = silicone
polymer or alcoholic wax
• Mobile Phase – inert carrier gas (Helium or Nitrogen)

ELECTROPHORESIS

❖ Involves the migration of charged solutes or particles in a supporting medium under the influence of an
electric field

• Iontophoresis – migration of small ions or molecules

• Zone electrophoresis – migration of charged macromolecules in a porous medium such as
cellulose acetate, paper or agarose
➢ it generates an ELECTROPHORETOGRAM – a display of protein zones

Principle:

• An ampholyte carries either a positive (+) or negative (-) charge; a zwitterion

• In an acid solution, an ampholyte receives protons and thereby carries a net (+) charge and migrates
towards the CATHODE
• In an alkaline solution, an ampholyte gives up protons and thereby carries a net (-) charge and migrates
towards the ANODE

Migration depends on:

1. Net electrical charge of molecule

2. Size and shape of the molecule
3. Strength of the electrical field
4. Properties of the support medium
5. Temperature of operation

STAINS: Protein Stains

o Amido Black
o Bromphenol Blue
o Coomasie Brilliant Blue
o Nigrosin

Page 66 of 120
 o Ponceau S

Isoenzymes: Nitrotetrazoleum Blue

Lipoprotein
o Fat Red 7B (Sudan Red)
o Oil Red O
o Sudan Black B

TYPES of ELECTROPHORESIS:

1. Paper Electrophoresis (PE)

2. Agarose Gel Electrophoresis (AGE)
3. Cellulose Acetate Electrophoresis (CAE)
4. Polyacrylamide Gel Electrophoresis (PAGE)
5. Starch Gel Electrophoresis
6. Isoelectric Focusing

ISOELECTRIC FOCUSING

⦿ An electrophoretic method in w/c proteins are separated on the basis of their pI (isoelectric pH)
⦿ Makes use of the property of proteins that their net charges are determined by the pH of their local
environment
⦿ Proteins show considerable variation in pI, but pI values fall in the range pH 3–12 (many having pIs
between pH 4–7)

1. Establishing pH gradients
Accomplished w/ the use of:
a. Carrier Ampholytes (Amphoteric electrolytes)
- Mixtures of molecules containing multiple aliphatic amino & carboxylate groups (buffer molecules)
- Included directly in IEF gels
b. Acrylamide buffers
- Derivatives of Acrylamide containing both reactive double bonds & buffering groups
- Covalently incorporated in PAG at the time of casting

2. Gel for Isoelectric Focusing

❖ Polyacrylamide Gel – large-pore convective matrices
- Polymerized with an initiator system including Riboflavin for photo-polymerization

AUTOMATION

HISTORY OF AUTOMATED ANALYZERS

⦿ 1957 – introduction of the 1st automated analyzer by Technicon

- Continuous-flow : sequential batch analyzer capable of providing single test result on approx. 40
samples / hour

⦿ Simultaneous Multiple Analyzer (SMA) – Technicon instruments w/c were next developed
- With multiple channels (for diff. tests)
- 6-12 test results simultaneously at the rate of 360 – 720 tests per hour
⦿ IMMUNOCHEMISTRY – specialty area w/ rapidly developing arsenal of analyzers
- Immunological techniques for assaying drugs, specific proteins, tumor markers & hormones
- Fluorescence Polarization Immunoassay

Page 67 of 120
 - Nephelometry
- Chemiluminescent Detection

BASIC APPROACHES OF AUTOMATED ANALYZERS

I. CONTINUOUS - FLOW
- Liquids (reagents, diluents & samples) are pumped through a system of continuous tubing
- Samples are introduced in a sequential manner, following each other through the same network
- Batch analysis can be used (e.g. large # of specimen in one run)

⦿ More sophisticated continuous flow anayzers

Use parallel single channels to run multiple tests on each sample (e.g. SMA & SMAC)
⦿ Major drawbacks: significant carry-over problems & wasteful use of continuously flowing reagents

II. CENTRIFUGAL ANALYSIS

- Uses the force generated by centrifugation to transfer & then contain liquids in separate cuvets for
measurement
- Capable of running multiple samples, one test at a time, in a batch
MAJOR ADV.: batch analysis (e.g. COBAS – Bio by Roche Diagnostics)

III. DISCRETE ANALYSIS

- Most popular & versatile
- Separation of each sample & accompanying reagents in a separate container
- Capability of running multiple tests one sample at a time OR multiple samples one test at a time
- Random access, stat capabilities

Page 68 of 120
 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Experiment No. 11
ELECTROPHORESIS

OBJECTIVE: At the end of the activity, the students are expected to:

1. Describe the components of an electrophoresis set-up;

2. State the working principle of electrophoresis;
3. Appreciate the importance of electrophoresis for the separation of serum proteins;
4. Properly perform serum protein electrophoresis

MATERIALS/EQUIPMENT: Horizontal Gel Electrophoresis Unit

PROCEDURE:
The instructor shall provide an orientation of the components of the horizontal gel electrophoresis set-up and provide
an overview of the procedure in serum protein electrophoresis.

DRAW AND LABEL: The components of the Horizontal Gel Electrophoresis Unit

GUIDE QUESTIONS:
1. State the working principle of electrophoresis.
2. Describe the use of electrophoresis in laboratory work.
3. Enumerate and describe the most common support media which are used for electrophoresis.
4. Describe the use of the following agents in electrophoresis. Give examples of each:
a. Buffers
b. Clearing agent
c. Stains

Page 69 of 120
 CHAPTER VI: CARBOHYDRATES

Desired learning outcomes:

At the end of this chapter, you should be able to:

A. Be familiar with the metabolism of carbohydrate.
B. To identify the different specimen considerations of Glucose Measurement.
C. Discuss the different glucose measurements and tests for diabetic patients.
D. To discuss the clinical significance of glucose.

I. BIOCHEMISTRY: Review of Carbohydrate Metabolism

II. SPECIMEN CONSIDERATIONS : Glucose Measurement
1. use serum, plasma or whole blood
2. whole blood values 10 - 15% lower than serum or plasma
3. serum or plasma should be refrigerated and separated from the cells within1 hour to prevent
substantial loss thru glycolysis
4. separated unhemolyzed serum at rm temperature is stable for 8 hr; ref temperature for 48
- 72 hr
5. preferred additive : sodium fluoride (gray top)
6. FBS : approximately 8 - 10 hr fast (not >16 hr)
7. CSF glucose : immediate processing to prevent bacterial utilization of glucose
8. urine sugar : only for monitoring of diabetics and not for diagnosis

III. GLUCOSE MEASUREMENT :

(1) Chemical methods : oxidation-reduction

− done in an alkaline medium
− glucose + boiling water -------- > eneidol (enol anion form) : reduce metallic ions

(a) Alkaline Copper reduction

1. Folin-Wu: phosphomolybdic acid ---------- > phosphomolybdenum blue
- total reducing substances (true reducing substances and saccharoids) measured
2. Nelson-Somogyi – arsenomolybdic acid ---------- > arsenomolybdenum blue
- barium sulfate enables measurement of true reducing substances only

3. Neocuproine – Campbell and King method

- neocuproine (7,9- dimethyl-1,10-phenanthroline) ----------- > cuprous-neocuproine complex
(yellow-orange)

(b) Alkaline Ferric reduction

− reverse colorimetric method
− ferricyanide ----- > ferrocyanide
− more specific than copper reduction
1. Johnson method
2. Folin/ Prussian blue method
3. Hagedorn-Jensen method

(2) Condensation methods

(a) with aromatic amines : Ortho-toluidine method by Dubowski (reference method)

- based on the ability of carbohydrates to form Schiff bases with aromatic amines
- glucose + ortho-toluidine ----> N-glycosylamine: Schiff base (green) 630 nm
- interfering substances: galactose, mannose and aldopentose

Page 70 of 120
 (b) with phenols :
− glucose ---> water + hydroxymethylfurfural (HMF)
− HMF + anthrone ---> green colored compound

(3) Enzymatic methods

(a) Glucose Oxidase method

− most specific enzyme reacting with only β-D-glucose

− glucose + O2 + H2O --- GOD ---> gluconic acid + H2O2

− H2O2 + reduced chromogen --- POD---> oxidized chromogen + H2O

mutarotase : added to convert alpha to beta glucose

− Methods to quantify H2O2 :

1. Gochman and Schmitz: 3-methyl-2-benzathiazolinone hydrazone + N,N-

dimethylaniline ---> indamine dye (590-66- nm)

2. Trinder: p-aminophenazone + phenol ---------- > purple product

(quinoneimine)

3. Miskieweis: 2,2-azine-di(3-ethyl bezothiazoline-(6)-sulfonic acid -------------- > colored chromogen

POLAROGRAPHIC OXYGEN ELECTRODE

- measures the amount of oxygen consumed in the glucose oxidase method
- To prevent the formation of oxygen from H2O2 (by catalase present in some GOD preparations), it is
removed by inclusion of two additional reactions:

❖ H2O2 + ethanol ----- catalase------ Acetaldehyde + H2O

❖ H2O2 + H+ + 2I- --- molybdate ---- I2 + 2H2O

Self-Monitoring of Blood Glucose: point of care device

- recommended for type 1 diabetics
- to maintain levels as close to normal as possible
- whole blood glucose testing using capillary blood as sample

(b) Hexokinase method

- more accurate, less interferences than glucose oxidase methods (REFERENCE method)
- not affected by uric acid and ascorbic acid
- false decrease: hemolyzed sample and elevated bilirubin levels
- can also be used for urine, CSF and serous fluids

− glucose + ATP ---hexokinase ---> glu-6-phosphate + ADP

− glu-6-phosphate + NADP ---G-6-PD ----------> NADPH + H + 6-phosphogluconate (340 nm)

(c) Glucose Dehydrogenase (GDH)

Glucose + NAD ------ GDH ----- Gluconolactone + NADH + H+ Coenzyme I:

NAD
Coenzyme II: NADP

Page 71 of 120
 IV. CLINICAL SIGNIFICANCE
1. HYPERGLYCEMIA
− increased plasma glucose levels
− caused by imbalance of hormones
− normally : insulin is secreted : enhance entry of glucose into the liver, muscle and adipose and
alters glucose metabolic pathways

Laboratory Findings in Hyperglycemia

1. increased glucose in plasma and urine
2. increased urine specific gravity
3. increased serum and urine osmolality
4. ketonemia and ketonuria
5. decreased blood and urine pH (acidosis)
6. electrolyte imbalance

CLASSIFICATION of DIABETES MELLITUS:

1. Type 1 DM (due to autoimmune β-cell destruction, usually leading to absolute insulin deficiency)
2. Type 2 DM (due to a progressive loss of β-cell insulin secretion frequently on the background of insulin
resistance
3. Gestational diabetes mellitus (GDM) – diabetes diagnosed in the 2nd or 3rd trimester of pregnancy that was
not clearly overt diabetes prior to gestation
4. Due to other causes:
- monogenic diabetes syndromes (e.g. neonatal diabetes & maturity-onset diabetes of the young [MODY]
- diseases of the exocrine pancreas (e.g. cystic fibrosis)
- drug- or chemical-induced diabetes (e.g. with glucocorticoid use)

CRITERIA for the DIAGNOSIS of DIABETES

FPG ≥ 126 mg/dL (7.0 mmol/L). Fasting is defined as no caloric intake for at least 8 hours
OR
2-H PG ≥ 200 mg/dL (11.1 mmol/L) during an OGTT. The test should be performed as described by the WHO, using
a glucose load containing the equivalent of 75 g anhydrous glucose dissolved in water.
OR
A1C ≥ 6.5% (48 mmol/mol). The test should be performed in a laboratory using a method that is NGSP certified and
standardized to the DCCT assay
OR
In a patient with classic symptom of hyperglycemia or hyperglycemic crisis, a random plasma glucose
≥200 mg/dL (11.1 mmol/L)

Reference: American Diabetes Association. Classification and diagnosis of diabetes. Sec 2. In Standards of Medical
Care in Diabetes – 2017. Diabetes Care 2017; 40(Suppl. 1):S11 – S24

Type 1 vs Type 2 Diabetes mellitus

Type 1 DM Type 2 DM
Frequency 5-10% 90-95%
Age of onset Any; most common in children and More common in advancing age; can
young adults occur in children and adolescents

Risk factors Genetic (HLA-DR/DQ on chr 6) Genetic

Autoimmune Environmental Obesity (BMI)
Sedentary lifestyle
Race/ethnicity

Pathogenesis Destruction of beta cells usually No autoimmunity

autoimmune Insulin resistance
Progressive insulin deficiency

C peptide Very low or undetectable Detectable

Page 72 of 120
 Pre-diabetes Autoantibodies Autoantibodies absent (testing not
(GAD65,ICA512,IAA) may be indicated)
present
GAD glutamic acid decarboxylase
ICA islet cell antigen
IAA insulin autoantibodies

Medication Insulin Oral hypoglycemic agents

therapy

Therapy to None known Lifestyle change

prevent or delay Oral hypoglycemic agents (metformin
onset of diabetes and acarbose)

TOLERANCE TESTS

⦿ PATIENT PREPARATION:
1. 3 days of unrestricted diet
2. Avoid medications that will interfere with the test
3. No beverages
4. No alcohol / no cigarettes
5. Overnight (8 – 14 hour) fast

ORAL GLUCOSE TOLERANCE TEST

A. 2-hour Postprandial Test

Dose: a gram / kg body wt.
Normal: peak value in 30 mins. (~150 mgs%); back to normal in 2 hrs.
B. 5-hour Postprandial Test Dose:
75 grams (WHO 1985) Normal: back
to normal in 5 hours
◾ Done only if value of 2-hr PPT is >140 – 160 mgs%

INTRAVENOUS GLUCOSE TOLERANCE TEST (IVGTT)

⦿ Indications:
◾ Poor absorption of ingested CHO
◾ History of GIT surgery
⦿ Dose: 0.5g/kg (25 g/dL solution given intravenously)

INSULIN TOLERANCE TEST

⦿ Dose: 0.1 unit / kg
⦿ Normal: In 30 mins, 50% decrease in FBS level; back to normal on 2nd hr.

Page 73 of 120
CLINICAL SIGNIFICANCE:
1. Insulin Resistance
❖ Delayed decrease in BGL
❖ DM, hyperadrenalism, Acromegaly
2. Hypoglycemic Unresponsiveness
❖ Normal fall of BGL but delay in regaining normal value
❖ Addison’s disease, hypofunction of anterior PG, hyperinsulinism, Von Gierke’s disease

TOLBUTAMIDE TOLERANCE TEST

⦿ Orinase or Tolbutamide
◾ Stimulates pancreas to produce insulin
◾ Evaluates hypoglycemia caused by insulinoma
⦿ Normal: In 30 mins, 50% decrease in FBS level; back to normal on the 2nd hour
EPINEPHRINE TOLERANCE TEST
⦿ Serves as an index of the quantity & availability of glycogen
⦿ Interpretation: Peak value after 30 mins.; normal within 2 hours
⦿ 35 – 45 mgs% increase in conc. between 45 – 60 mins.
LACTOSE TOLERANCE TEST
⦿ Detects the presence of mucosal lactase
⦿ Causes of Lactose Intolerance:
◾ Deficiency of lactase
◾ Inhibition to lactase activity (e.g. intestinal disorders)
GESTATIONAL DIABETES MELLITUS
Defined as any degree of glucose intolerance that was first recognized during pregnancy, regardless of whether
the condition may have predated the pregnancy or persisted after the pregnancy

2. HYPOGLYCEMIA
- results from an imbalance between glucose utilization and production
- observable symptoms appear at about 50-55 mg/Dl
- Symptoms :
A. Neurogenic
- triggered by the ANS
- tremulousness, palpitations, anxiety
- diaphoresis, hunger, paresthesias
B. Neuroglycopenic
- CNS hypoglycemia
- dizziness, tingling, difficulty concentrating, blurred vision
- confusion, behavioral changes, seizure, coma

* historically : postabsorptive (fasting) and postprandial (reactive) hypoglycemia

TESTS FOR MONITORING DIABETIC PATIENTS

A. Measure glycosylated hemoglobin (HbA1c) every 3-6 months

- glucose + amino group of hemoglobin = ketoamine (rate of formation is directly proportional to plasma
glucose concentration)
- provides an index of average BGL over the past 2-4 months
- EDTA whole blood sample
◾ Values for total HbA1 & HbA1c have a high degree of correlation

Page 74 of 120
METHODS for MEASUREMENT of Hemoglobin A1C:
1. ION-EXCHANGE CHROMATOGRAPHY
⦿ Cation-exchange resin or carboxymethyl cellulose resin
2. HPLC
Reference method
3. COLORIMETRY
⦿ Hb A1c --- acid------ 5-HMF
4. RADIOIMMUNOASSAY (RIA)
⦿ Antibodies against Hb A1c (sheep antiserum)
5. ELECTROPHORESIS
⦿ Citrate agar electrophoresis (pH 6.0-6.2)
⦿ Good resolution of Hb A & Hb A1
6. ISOELECTRIC FOCUSING
⦿ Scanned on high-resolution microdensitometer
⦿ Hb A1c is adequately resolved from HbA1a, A1b, S and F
7. AFFINITY CHROMATOGRAPHY
⦿ Use of affinity gel columns

B. Microalbuminuria
- useful to assist in diagnosis at an early stage and before the development of proteinuria (> 0.5g/day)
C. C peptide
- reflects pancreatic secretion of insulin
- proinsulin cleaved to give C peptide and insulin
- reflects endogenous insulin production if patient is taking insulin

DETECTION OF ACUTE CRISIS SITUATIONS

Ketone measurement
− ketone bodies : produced by the liver thru the metabolism of fatty acids to provide a ready energy
source from stored lipids at times of low carbohydrate availability
− 3 types : acetone (2%), acetoacetic acid (20%) and 3-beta-hydroxybutyric acid (78%)
− ketonemia and ketonuria
− type 1 DM : acute illness, stress, pregnancy, hyperglycemia of >300mg/dL or with signs of
ketoacidosis
− DKA : serious and potentially fatal hyperglycemic condition
− nausea, vomiting, abdominal pain, electrolyte disturbances and severe
dehydration
− specimen considerations :
− fresh serum or urine
− tightly stoppered and assayed ASAP
− lab methods :
a. Gerhardt's : acetoacetic acid + ferric chloride ---------- > red color
b. acetoacetic acid + sodium nitroprusside ----------- > purple color
c. enzymatic method: acetoacetic acid + NADH + H ---β-HBDH ------------ > NAD + beta-
hydroxybutyric acid

Page 75 of 120
 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Experiment No. 12
BLOOD GLUCOSE DETERMINATION

Reference Ranges: Conventional Unit: 70 – 105 mg/dL

S.I. Unit: mmol/L
Conversion factor: 0.055

Method: (TRINDER/GOD)
Principle:
The enzyme glucose oxidase catalyzes the oxidation of glucose to gluconic acid and H2O2. The H2O2
reacts with phenol and 4-aminoantipyrine in the presence of peroxidase to form a quinoneimine dye. The intensity of
the color formed (pink) is proportional to the glucose concentration and can be measured photometrically between
480 and 520 nm.

PROCEDURE:
BLANK STANDARD SAMPLE

Glucose Reagent 1.0 ml 1.0 ml 1.0 ml

water 10 μL - -

Standard reagent - 10 μL -

Serum - - 10 μL

 Mix, incubate at 37 C for 5 minutes

 Read absorbance of standard (As) and samples (Ax) against reagent blank

Computation of results:

A (patient) X 100 mg/dL (Glucose Standard) = patient conc. (mg/dL) A

(standard)

Patient conc. (mg/dL) x 0.055 = Patient conc. (mmol/L)

Specimen / Stability: Serum, plasma, urine, CSF

Separated and non-hemolyzed samples are stable 8 hours at 25 C and 3 days at 2 – 8 C. Variable stability
is observed with longer storage periods. Glycolysis decreases serum glucose by approximately 5 – 7% in 1 hour (5-10
mg/dL) in normal uncentrifuged coagulated blood at room temperature. The rate of in- vitro glycolysis is higher in the
presence of leukocytosis or bacterial contamination. Glycolysis can be inhibited and glucose stabilized for as long as
3 days at room temperature by adding sodium iodoacetate or sodium fluoride (NaF) to the specimen.

Linearity: The method is linear for glucose values up to 500 mg/dl.

GUIDE QUESTIONS:
1. Differentiate the types of Diabetes mellitus as provided by the American Diabetes Association.
2. Present the criteria which is used for the diagnosis of Diabetes mellitus.

Page 76 of 120
3. Describe the test strips used for monitoring blood glucose.
4. Provide an association between diabetes mellitus and hypertension.
5. Discuss the pathogenesis of Diabetic Ketoacidosis (DKA).

Page 77 of 120
 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Experiment No. 13
GLYCOSYLATED HEMOGLOBIN

OBJECTIVES: At the end of the activity, the students are expected to:

1. Describe the mechanism by which glycosylated hemoglobin is formed;

2. Perform the procedure for the laboratory determination of glycosylated hemoglobin;
3. Discuss the importance of determining glycosylated hemoglobin in the patient’s blood sample;
4. Cite the principle involved in the test performed for glycosylated hemoglobin.

Reference Ranges:
NGSP IFCC
Prediabetes 5.7 – 6.4% 39 – 46 mmol/mol
Presence of diabetes ≥6.5% ≥48 mmol/mol
Target in diabetes <7.0% <53 mmol/mol

Method: Boronate Affinity Assay

Principle: The CERA-STATTM 4000 is a boronate affinity assay. The CERA-STATTM HbA1c Test Kit consists of the
test device, the R1 reagent and the R2 reagent. The R1 reagent contains the agents that lyse erythrocytes and
precipitate hemoglobin specifically, as well as a blue boronic acid conjugate that binds cis-diol of glycated
hemoglobin.

When blood is added to the R1 reagent, the erythrocytes are lysed and all hemoglobin precipitates. The
boronic acid conjugate binds to the cis-diol configuration of glycated hemoglobin. An aliquot of the reaction mixture is
added to the test device and all the precipitated hemoglobin, conjugate- bound and unbound, remains on top of the
filter. Any unbound boronate is removed with the R2 reagent. The precipitate is evaluated by measuring the blue
(glycated hemoglobin) and the red (total hemoglobin) color intensity respectively with the CERA-STATTM 4000
Analyzer, the ratio between them being proportional to the percentage of glycated hemoglobin in the sample.

Procedure:
1. Perform a clean capillary puncture. Fill the capillary tube provided in the reagent kit with blood.
2. Place the filled capillary tube into the vial which contains reagent 1 (R1). Mix ten (10) times by gentle inversion
and allow the vial to stand for two (2) minutes.
3. After gently mixing again the contents of the vial, use an automatic pipet to aspirate 25μL of the mixture and
transfer this into the reaction area of the cartridge. Allow the sample to be absorbed into the membrane for at least 10
seconds.
4. Add 25μL of reagent 2 (R2) into the test device. Again, allow sample to be absorbed into the
membrane for at least 10 seconds.
5. Insert the test device into the tray of the CERA-STATTM 04000 Analyzer and record the %HbA1c and estimated
blood sugar level as indicated on the screen.

GUIDE QUESTIONS:
1. What is glycosylated hemoglobin?
2. Describe three (3) methods for its laboratory determination.
3. Describe the methods employed for the diagnosis of Gestational Diabetes Mellitus as proposed by ADA and
NIH.

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 CHAPTER VII: PROTEINS

Desired learning outcomes:

At the end of this chapter, you should be able to:
A. Discuss the structure and physiology, classifications, functions, and metabolism of proteins.
B. Identify and Discuss the different major plasma proteins and their clinical significance.
C. Identify the methods of protein measurement and their principles.

BIOCHEMISTRY: Structure & Physiology

-covalently linked polymers of amino acids
- amino acids are linked via peptide bond (formed upon removal of a water molecule)
- amphoteric
- synthesized in the liver & secreted by the hepatocyte into the circulation EXCEPT
immunoglobulins (plasma cells)
- provide 12 – 20% of the total daily body energy requirement
- composes 50 – 70% of the cell’s dry weight

1. Primary structure – sequence of the amino acids in the polypeptide chain

− - analytical processes: chromatography, electrophoresis, dye binding and light absorbance depend
on this sequence
2. Secondary structure – arises from the interaction among the different segments of a polypeptide
chain
Three structures:
a. alpha-helix – chain forms a regular helix ; coil resembling a spring
b. beta-pleated sheets – in fully extended structures; flat, corrugated structure
c. random coils – no apparent pattern
3. Tertiary structure – actual 3-dimensional structure or folding pattern of the protein
4. Quaternary structure – association of several polypeptide chains into larger “oligomeric” aggregate unit
- stable complexes: dimers, trimers, tetramers

Classification

1. Simple – contain peptide chains that on hydrolysis yield only AA

a. globular: relatively symmetrical with compactly folded and coiled polypeptide chains
b. fibrous: more elongated and asymmetrical and have a higher viscosity
2. Conjugated – protein (apoprotein) + nonprotein (prosthetic group)
prosthetic group : lipid (lipoprotein) , carbohydrate (glycoprotein) , metals (metalloprotein) , etc.

Functions
- Repair body tissues
- Important in blood coagulation and immunologic function
- For transport of metabolic substances
- Maintenance of osmotic pressure
- Maintenance of blood pH (buffers)
- Biocatalysts

Metabolism
1. Dietary Intake
2. Absorption
3. Production
4. Storage
5. Destruction

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MAJOR PLASMA PROTEINS

1. PRE-ALBUMIN (Transthyretin)
− Migrates ahead of albumin
− Rich in tryptophan and contain 0.5% carbohydrate
− Serves as transport protein for T3, T4 and retinol (Vit. A)
− Increased: alcoholism, chronic renal failure, steroid treatment
− Decreased: poor nutrition, liver disorder, malignancy

2. ALBUMIN
− largest plasma protein fraction (52-62%)
− synthesized in the liver at a rate that is dependent on protein intake
− serves as circulating reservoir of amino acids
− regulator of osmotic pressure
− transport protein because of ease of binding with blood components
− a “negative acute phase reactant”
− sensitive & highly prognostic marker in cases of cystic fibrosis
• Reference values: 3.5 – 5.0 g/dL (35 – 50 g/L)
− Increased albumin (Hyperalbuminemia):
− hemoconcentration, dehydration
− excessive albumin infusion
− Decreased albumin (Hypoalbuminemia):
− decreased synthesis (liver impairment)
− malabsorption or malnutrition
− nephrotic syndrome (renal loss)
− severe burns
3. GLOBULINS – heterogenous complex mixture of protein molecules (α1, α2, β and γ fractions)
- Elevated concentration of globulin in early cirrhosis will balance loss of albumin resulting to normal
levels of total protein
- normal A/G ratio : 1.3 – 3 : 1
- low A/G ratio – liver diseases, infectious diseases, multiple myeloma, nephritis

A. ALPHA 1 GLOBULINS

a. α-1-antitrypsin – 90% of the α1-globulin band

- an acute phase reactant
- neutralize trypsin-like enzymes
- major inhibitor of protease activity; inhibit lysosomal elastase released from PMNs during their
response to particles & inhaled bacteria
- Increased: inflammation, pregnancy and contraceptive use
- Decrease: associated with emphysematous pulmonary disease & juvenile hepatic cirrhosis

b. α-1-fetoprotein – synthesized initially by the fetal yolk sac & then the parenchymal cells of the liver
- used as a tumor marker (hepatic & gonadal CA)
- screening test for any fetal conditions, increase passage of fetal proteins into the amniotic fluid;
detects neural tube defects
- Increased: neural tube defects (spina bifida), atresia of the GIT, fetal distress, ataxia
telangiectasia, tyrosinosis, hemolytic disease of the newborn (HDN)
- Decreased: Down's syndrome

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 c. α-1-Acid Glycoprotein (orosomucoid)
- contains high percentage of CHO and sialic acid (45% CHO + 11-12% Sialic acid)
- synthesized both by the liver & by granulocytes and monocytes
- inhibits the phagocytic activity of neutrophils & inhibits platelet aggregation
- may inactivate progesterone
Increased: pregnancy, cancer, pneumonia, rheumatoid arthritis (RA), cell proliferation
Decreased: nephrotic syndrome

d. α-1-Antichromotrypsin- a member of the serine proteinase inhibitor (serpin) family.

- produced in the liver
- an acute phase protein
- it inhibits the activity of enzymes cathepsin G, pancreatic elastase, mast cell
chymase, and chymotrypsin by cleaving them into different shapes.

B. ALPHA 2 GLOBULINS

1. Haptoglobin
✓ an acute phase reactant
✓ synthesized in the hepatocytes & cells of the RES
✓ binds free hemoglobin by its α- chain
✓ one of the proteins used to evaluate rheumatic diseases
Increased: inflammation, nephrotic syndrome, burns, trauma
Decreased: intravascular hemolysis and liver disease

2. Ceruloplasmin
✓ copper-containing protein BUT does NOT transport copper; synthesized in the liver, where 6-8 copper
atoms are attached
✓ serves as an antioxidant to prevent lipid peroxidation & cellular damage
✓ increased in inflammation and pregnancy
✓ indicator for Wilson's disease (0.1 g/L)
- Decreased: Wilson’s disease, malnutrition, malabsorption, nephrotic disease, Menke’s disease
(kinky hair syndrome)

3. α-2-Macroglobulin – largest major non-immunoglobulin protein in plasma

- found principally in the intravascular spaces; does not diffuse from the plasma space
- lower amounts can also be found in CSF
- inhibits proteases such as trypsin, pepsin and plasmin

C. BETA GLOBULINS

a. Transferrin (Siderophilin)
- major component of the β2-globulin fraction
- a glycoprotein synthesized in the liver
- transports oxidized iron (Fe+3)to its storage sites
- prevents loss of iron through the kidney
Increased: hemochromatosis (bronze skin)
Decreased: liver disease, malnutrition, nephrotic syndrome

b. β2-Microglobulin
- light chain component of the major human leukocyte antigen (HLA)
- found on the surface of most nucleated cells
- present in high concentration on lymphocytes
- filtered by the renal glomerulus but reabsorbed
Increased: RA, systemic lupus erythematosus (SLE), HIV

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 D. GAMMA-GLOBULINS
- Increased in:
• chronic inflammation
• cirrhosis or viral hepatitis
• collagen diseases
• paraproteins (monoclonal bands, gammopathies)
- Decreased in congenital or acquired immunodeficiency

Immunoglobulins – synthesized in the plasma cells

- not produced to any extent by the neonate

E. FIBRINOGEN – one of the largest proteins in the blood plasma

- synthesized in the liver
- most abundant of the coagulation factors
- an acute phase reactant; markedly increased during inflammatory process
- may serve as a marker for long-term prognosis of cardiovascular disease
- distinct band between β and γ-globulins on electrophoresis
- it forms a fibrin clot when activated by thrombin
Increased: pregnancy and use of birth control pills
Decreased: extensive coagulation
Reference values: 200 – 400 mg/dL (2.0 -4.0 g/L)

F. LIPOPROTEIN
- complexes of proteins & lipids (LDL, HDL, VLDL, Chylomicrons)
- transports cholesterol, triglyceride and phospholipids

G. COMPLEMENT
- participates in the immune reaction and serve as a link to the inflammatory response
- circulates as non-functional precursors
Increased: inflammatory states Decreased:
SLE, DIC, malnutrition

G. C-REACTIVE PROTEIN
- appears in blood of patients with diverse inflammatory diseases
- undetectable in healthy individuals
- precipitates with the C substance, a polysaccharide of pneumococci
- general scavenger molecule; gamma-migrating protein

Increased: acute rheumatic fever, MI, RA, gout, bacterial & viral infections

MISCELLANEOUS PROTEINS:

1. Myoglobin
▪ A heme protein or oxygen-binding transport protein found in skeletal and cardiac muscles
▪ Approximately 2% of the total muscle protein
▪ Marker for chest pain (angina) and early detection of acute myocardial infarction (AMI)
▪ In AMI, the onset is 1-3 hours, peak level 5-12 hours, normalize in 18-30 hours
▪ Useful marker for monitoring the success or failure of reperfusion
Increased: AMI, angina, rhabdomyolysis, muscle trauma, extrenuous exercise, IM injection
2. Troponins
▪ A complex of 3 proteins that bind to the thin filaments of striated muscle (cardiac & skeletal)
▪ Diagnostic marker for identifying cardiac injury in the presence of a skeletal muscle damage
▪ Levels in blood may elevate after AMI in the absence of CK-MB elevations
▪ TnT, TnI, TnC = muscle contraction

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3. Amyloid
▪ A pathological extracellular deposit associated with a group of disorders collectively called
amyloidosis.

MEASUREMENT OF PROTEINS

1. KJELDAHL METHOD
▪ determination of protein nitrogen derived from constituent amino acids
▪ Nitrogen in pff is converted to ammonia using H2SO4
⦿ The nitrogen in ammonia may be measured using:
◾ Nesslerization
◾ Berthelot reaction
◾ Titration method

2. BIURET METHOD
▪ Copper binds to the peptide bond structure of the protein, forming a purple-colored chromogen

3. FOLIN-CIOCALTEU METHOD
▪ Tyrosine & tryptophan in proteins reduce PT-PMA reagent (folin-ciocalteu) to give a blue color
▪ Detects proteins in concentrations as low as 10 – 60 µg/mL
▪ Widely used in research to measure tissue & enzyme proteins

4. Absorption of UV light at 280 nm

▪ Chiefly the result of the high electron density of aromatic rings of tyrosine & tryptophan in solution (pH 8)

5. Dye-Binding Methods
▪ Based on the ability of proteins to bind certain dyes
▪ Bromcresol Green Method (BCG) for Albumin
▪ Used to stain protein bands after electrophoresis (e.g. Coomassie Brilliant Blue)

7. PROTEIN ELECTROPHORESIS
▪ direction of migration of proteins in an electrical field determined by surface charge & MW of protein
▪ Urine protein electrophoresis exactly same as serum except it must be concentrated before
application
▪ Support media include cellulose acetate, agarose gel and starch gel
▪ Stains: amido black, ponceau S and coomassie brilliant blue

SIGNIFICANT FINDINGS:
❖ Beta-gamma bridging effect: due to IgA (in serum); Cirrhosis
❖ Monoclonal band (gamma-globulin): Monoclonal gammopathy (Multiple myeloma)
❖ Polyclonal band: Chronic inflammation
❖ Increase in α-2-macroglobulin: nephrotic syndrome
❖ α-1-globulin flat curve: deficiency in α-1-antitrypsin; Juvenile cirrhosis

AMINO ACIDURIA
1. Overflow - plasma level above renal threshold as a result of metabolic disorder
a. Phenylketonuria (PKU) – enzyme deficiency (phenylalanine hydrolase) which leads to :
- increased phenylalanine in the blood
- phenylpyruvic acid (prime metabolite) is present in both blood and urine in elevated
concentration

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 b. Branched chain ketoaciduria – maple syrup urine disease
- caused by markedly reduced or absence of α-ketoacid decarboxylase
- increased branched chain amino acids (leucine, isoleucine & valine) in blood, urine and CSF

2. Renal - normal plasma level but decreased renal threshold or reabsorption

• cystinuria – increased cystine, lysine, ornithine, arginine in urine

Methods
1. Screening tests
a. TLC with ninhydrin
b. urine color tests
c. Guthrie microbiologiocal tests : PKU

2. Quantitative tests
a. Ion-exchange chromatography
b. HPLC
c. GC-MS

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 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Experiment No. 14
ALBUMIN, TOTAL SERUM PROTEINS, A/G RATIO

OBJECTIVES: At the end of the activity, the students are expected to:

 Correlate test results with pathologic conditions given an electrophoresis pattern, identify and correlate abnormal
result with pathologic findings;
 Compute for the A/G ratio and interpret test results with pathologic conditions;
 Explain the process of amino acid synthesis and metabolism;
 Discuss the different types of aminoacidopathies.
 Consistently maintain quality control in patient preparation, specimen collection, sample processing & handling;
 Appreciate the significance of understanding how metabolism influences the values of various analytes in blood.
 Perform correctly laboratory methods for total protein & serum albumin determination.

I. Serum Albumin Determination:

Method: Bromcresol Green (BCG) Method

Principle:
Determination of Albumin in serum or plasma is usually based on the binding behavior of the
protein with an anionic dye, bromcresol green forming a green colored complex.

COLORIMETRIC PROCEDURE
BLANK STANDARD SAMPLE

Reagent 3.0 mL 3.0 mL 3.0 mL

water 20 μL - -

standard - 20 μL -

serum - - 20 μL

1. Mix well by gently inversion and stand at room temperature for 1 min.
2. Read absorbance at 600 nm against the reagent blank.

Stability: 60 mins.

II. Determination of Total Proteins:

Method: Biuret, Modified
Principle:

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 When a solution containing proteins is rejected with a tartrate-complex cupric ions and alkali, the
copper binds to the peptide bond structure of the proteins, forming a purple colored chromogen.

COLORIMETRIC PROCEDURE
BLANK STANDARD SAMPLE

Protein Reagent 3.0 mL 3.0 mL 3.0 mL

water 20 μL - -

standard - 20 μL -

serum - - 20 μL

1. Mix well by gently inversion. Stand at room temperature for 5 mins.

2. Read absorbance at 550 nm against a reagent blank.

Stability: 60 mins.

III. Determination of Globulin:

Globulin = gms. of total protein - gms. of albumin

IV. A/G RATIO:

A/G Ratio = gms. of albumin

gms. of globulin

NORMAL VALUES AND CONVERSION FACTORS

CONVENTIONAL C.F. S.I. (gms%)

(gms%)
Total proteins 6-8 10 60 - 80

Albumin 3.5 - 5 10 35 – 50

Globulin 1.8 - 3.2 10 18 - 32

A/G ratio 1.5-2.4 : 1

GUIDE QUESTIONS:
1. Describe the different functions of proteins.
2. In tabular form, illustrate the structures of the different immunoglobulins and give the function(s) of each.
3. What are the functions of pre-albumin and acute phase reactants?
4. Give the principle underlying serum protein electrophoresis.
5. Illustrate the migration pattern of proteins in serum.

Page 86 of 120
 CHAPTER VIII: NON-PROTEIN NITROGEN COMPOUNDS

Desired learning outcomes:

At the end of this chapter, you should be able to:

A. Discuss the different functions of the kidneys.
B. Elaborate and discuss the different Non-Protein Nitrogen Compounds ( Urea, Creatinine, Uric Acid, Ammonia)
C. Identify the clinical significance of the the different Non-Protein Nitrogen Compounds.
D. Discuss the different NPN determination.

FUNCTIONS of the KIDNEYS:

1. Elimination of excess body water
2. Elimination of waste products of metabolism (urea and creatinine)
3. Elimination of foreign substances like drugs
4. Retention of substances necessary for normal body function (proteins & amino acids, glucose)
5. Regulation of electrolyte balance and osmotic pressure of the body fluids)
6. Endocrine function:
Primary: production of rennin, prostaglandin and erythropoietin Secondary:
degradation of insulin, glucagon and aldosterone

Clinically Significant NPN compounds:

1. urea – 45%
2. amino acids – 20%
3. uric acid – 20%
4. creatinine – 5 %
5. creatine – 1-2 %
6. ammonia – 0.2%

TOTAL NPN METHODOLOGY:

TWO STEPS:
1. KJELDAHL DIGESTION
 The nitrogen in a pff of the specimen is converted to ammonia using hot conc. H2SO4 with copper sulfate,
mercuric sulfate or selenium oxide as the catalysts.

NPN + H2SO4 NH4HSO4

NH4HSO4 + NaOH Na2SO4 + NH3 + H2O

2. MEASUREMENT OF AMMONIA FORMED

A. NESSLERIZATION
Nessler’s reagent – double iodide salt of potassium & mercury Gum
ghatti – colloidal stabilizer
Dimercuric ammonium iodide – yellow to orange brown product NH3

+ HgI2.2KI NH2Hg2I2 + KI + NH4I

B. BERTHELOT METHOD
Reagent: phenol and alkaline hypochlorite Catalyst:
sodium nitroprusside
Product: indophenol blue

NH3 + NaOCl + Phenol Indophenol + NaCl + H2O

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 C. MONITORING CONSUMPTION OF AMMONIA (Kaplan, Manoukian – Fawaz; Kallet – Cook
Reaction)

NH3 + α – ketoglutarate + NADH + H Glutamate + NAD Catalyst:

Glutamate dehydrogenase
• Measure a decrease in the absorbance at 340 nm

UREA

Biochemistry
- most abundant NPN compound in plasma
- major excretory product of protein metabolism
- synthesized in the liver from CO2 and ammonia that arises from the deamination of amino acids in the
reaction of the urea cycle
STRUCTURE: NH2- C = O

NH2
MW = 60 g/mole where: C = 1 x 12 = 12
H=4x1=4
O = 1 x 16 = 16
N = 2 x 14 = 28
Conversion factors:
From urea mass units to urea nitrogen (28/60) = 0.467 From
urea nitrogen to urea mass units (60/28) = 2.14 Renal handling:
- 90% excreted through the kidneys
- 10% excreted through the skin and GIT
- 40 – 70% reabsorbed in the renal tubules by passive diffusion
Urea concentration depends on the following :
1. renal function and perfusion
2. protein content of the diet
3. amount of protein catabolism

METHODS FOR UREA DETERMINATION

I. INDIRECT METHOD / ENZYMATIC = measures Blood Urea Nitrogen
- Based on the preliminary hydrolysis of urea with urease followed by some process that
quantitates the ammonium ion

1. Berthelot reaction
2. Nessler’s reaction
3. GLDH-coupled enzymatic method (Dupont ACA Analyzer)
➢ Decrease in absorbance of NAD at 340 nm
4. Conductimetric method: Beckman BUN Analyzer
➢ Based on the measurement of the conductivity generated from the reaction of urease on urea
producing ammonium ions & bicarbonates
5. Urograph or Urastrat strip
➢ Physical principle: based on chromatography
➢ Chemical principle: Conway Microdiffusion method
6. Indicator dye (uriol): Kodak Ectachem Analyzer
➢ Dye is added to NH4 ions from urea hydrolysis & the color change is measured
➢ Used in multilayer film reagents, dry reagent strips and automated systems

Page 88 of 120
 II. DIRECT METHODS
1. Diacetyl Monoxime (Fearon)
➢ Direct condensation reaction
➢ Diacetyl – very toxic

2. ortho – phthaldehyde: adapted by automated methods Urea

+ OP Isoindoline

Isoindoline + Naphthylethylenediamine colored compound

DISEASE CORRELATIONS:

1. Azotemia – a biochemical abnormality pertaining to increase NPN compounds especially creatinine and urea
defining GFR defect

Three categories:
a. prerenal – due to reduced renal blood flow
> hemorrhage, dehydration, increased protein catabolism
b. renal – decreased renal function
> kidney diseases: glomerular nephritis
c. postrenal – obstruction of urine flow
> calculi, tumors of bladder or prostate

2. Uremia/ Uremic syndrome – a clinical syndrome characterized by increased BUN accompanying

renal failure seen in metabolic acidosis, hyperkalemia and edema

3. Decreased Urea:
- decreased protein intake, severe vomiting and diarrhea

Specimen Requirements and Interfering Substances

1. plasma, serum or urine
2. plasma : ammonium ions and high concentrations of sodium citrate and sodium fluoride must be
avoided
3. non fasting sample is acceptable
4. nonhemolyzed sample is recommended
4. urine sample guarded against bacterial decomposition of urea

Reference Interval:

adult serum/plasma 6-20 mg/dL 2.1-7.1 mmol/L

urine, 24hr 12-20 g/day 0.43-0.71 mol/day

conversion factor : mg/dL ---> mmol/L : 0.357

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 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Experiment No. 15
BLOOD UREA NITROGEN DETERMINATION

OBJECTIVES: At the end of the activity, the students are expected to:

 Discuss the sources, metabolism, formation of Urea;

 Discuss the different factors that may affect the level of Urea in blood;
 Describe the principle involved, advantages of the commonly used methods for Urea determination;
 Recognize the effect of age, gender and other factors on the value of BUN;
 Consistently maintain quality control in patient preparation, specimen collection, sample processing & handling;
 Appreciate the significance of understanding how metabolism influences the values of various analytes in blood.
 Perform accurately assays for BUN.

Reference Ranges: Conventional Unit: 10-20 mg/dL

S.I. Unit: 1.7 – 8.3 mmol/L
Conversion factor: 0.357

Method: Coupled enzyme reaction (Urease & Glutamate DH)

Principle:
The urease enzyme hydrolyzes urea in the sample to release ammonium ions, which react with 2-
oxoglutarate and NADH in the presence of glutamate dehydrogenase to form glutamate and NAD. The decrease in
absorbance is measured at 340 nm.

Procedure:
BLANK STANDARD SAMPLE
Working Reagent 2.0 mL 2.0 mL 2.0 mL
Incubate at 37 C for 5 minutes
water 20 μL - -
standard - 20 μL -
serum - - 20 μL

 Mix; incubate 30 seconds at 37 C, then record absorbance as A1. After exactly 60 seconds, record again
absorbance as A2.

Computation of results:
Urea mg/dL = A2 – A1 (sample) x 50 (standard value)
A2 – A1 (standard)
Linearity: method for urea nitrogen is linear to values up to 300 mg/dL

Interferences:

Plasma collected in anticoagulant containing either ammonium or fluoride salts cannot be used. No
interference was observed by the presence of:

Page 90 of 120
Hemoglobin ≤500 mg/dL
Bilirubin ≤4.4 mg/dL
Lipids ≤600 mg/dL

GUIDE QUESTIONS:
1. Discuss the different functions of the kidney.
2. Illustrate the metabolism of Urea (Kreb’s-Henseleit Cycle).
3. Describe the following renal disorders:

a. Acute nephrotic syndrome

b. Rapidly progressive glomerulonephritis (RPGN)
c. Chronic glomerulonephritis
d. Acute renal tubular disease
e. Nephrolithiasis

4. What is the clinical significance of determining the BUN:Creatinine ratio?

Page 91 of 120
 CREATININE

Biochemistry
- principal waste product of muscle metabolism derived mainly from Creatine (alpha-
methylguanidinoacetate)
- Creatine is produced from two enzymatic processes:
o transamination of arginine & lysine forming guanidinoacetic acid in the kidneys, small
intestines, pancreas and probably the liver
o methylation of guanidinoacetic acid in the liver

Figure 1: PHOSPHORYLATION OF CREATINE IN SKELETAL MUSCLES

o Creatine PO4 is the muscles’ energy source

Figure 2: NON-ENZYMATIC CYCLIZATION OF EITHER CREATINE OR CREATINE PHOSPHATE FORMS

CREATININE

RENAL HANDLING of CREATININE:

1. Glomerular filtration
2. Excreted without being reabsorbed. Thus, excretion is relatively constant. Creatinine output is
sometimes used to measure the completeness of a 24-hour urine sample collection
3. When serum creatinine is elevated, it is secreted in the renal tubules

Page 92 of 120
ANALYTICAL METHODS

1. DIRECT METHOD: JAFFE REACTION

Creatinine + alkaline picrate Creatinine picrate (red orange/yellow)

510 nm

Alkaline picrate: 1 part 10% NaOH and 5 parts sat. picric acid (2,4,6 trinitrophenol)

NOTE:
• The Jaffe reaction lacks specificity
• Non-creatinine Jaffe-reacting chromogens:
- Proteins
- Glucose
- Ascorbic acid
- Guanidine
- Acetone
- Cephalosporins
- α-ketoacids (acetoacetate and pyruvate)

2. INDIRECT / ENZYMATIC METHODS

a. F. Lim – Creatininase or creatinine iminohydrolase

Creatinine ---------------- N-methylhydantoin + NH3

(Creatininase)

NH3 + α-ketoglutarate + NADH ----------------- glutamate + NAD + H+

(Glutamate DH)

b. G.A. Moss – Creatinine Amidohydrolase Creatinine ------

----------------------------- Creatine
(Creatinine amidohydrolase)

Creatine + ATP ------------------ CreatinePO4 + ADP

(Creatine kinase)

ADP + PEP ------------------ Pyruvate + ATP

(Pyruvate kinase)

Pyruvate + NADH + H+ --------------- Lactate + NAD

(Lactate DH)

3. YATZIDIS METHOD
 Creatinine reacts with alkaline picrate at two different pH levels
pH 10: protein & other interfering materials will reacts w/ picrate but creatinine does not pH 11: both
creatinine & proteins react

4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Sources of error :
1. ascorbate, glucose, alpha keto acids and uric acid – false increase
2. drugs : cephalosporin and dopamine intake – false increase
: lidocaine intake – false increase

Page 93 of 120
Specimen Requirements and Interfering Substances
3. plasma, serum or urine
4. avoid hemolyzed and icteric sample especially for the Jaffe reaction
5. lipemic samples cause errors
6. non-fasting sample acceptable
7. high protein ingestion may transiently elevate serum concentrations
8. urine : refrigerated after collection or frozen if longer storage than 4 days is required

DIAGNOSTIC APPLICATION:
− Creatinine Clearance
3. overestimates but gives a reasonable approximation of glomerular filtration rate

Creatinine Clearance = UxV

P
4. expressed in mL/minute
5. plasma concentration of creatinine is inversely proportional to creatinine clearance (elevated creatinine
clearance = decreased GFR)
6. insensitive marker: >50% renal dysfunction = abnormal plasma creatinine
Creatine kinase
used in the diagnosis of muscle diseases

BUN : CREATININE RATIO

NORMAL: 10–20:1

1. BUN:CREA ratio <10:1

a. Acute tubular necrosis
b. Low protein intake; starvation
c. Severe liver disease
d. Repeated dialysis
e. Severe vomitting or diarrhea

2. BUN:CREA ratio >10:1 with normal creatinine

a. Catabolic states w/ tissue breakdown
b. Pre-renal azotemia
c. High protein intake
d. After GIT hemorrhage

3. High ratio with elevated creatinine levels

✓ Post-renal obstruction
✓ Pre-renal azotemia superimposed on renal disease

NOTE: Creatinine determination is MORE SPECIFIC for the diagnosis of renal disease
BUN determination is MORE SENSITIVE for the diagnosis of renal disease

ASSOCIATED MYOPATHIES:
− muscular dystrophy
− familial periodic paralysis
− myasthenia gravis
− dermatomycosis

Page 94 of 120
Reference Interval
(1) plasma/serum
5. Jaffe adult female 0.6-1.1 mg/dL (53-97 µmol/L)
adult male 0.9-1.3 mg/dL (80-115 µmol/L)

6. Enzymatic adult female 0.5-0.8 mg/dL (44-71 µmol/L)

adult male 0.6-1.1 mg/dL (53-97 µmol/L)

(2) 24h urine

adult female 600-1800 mg/day 5.3-15.9 mmol/day
adult male 800-2000 mg/day 7.1-17.7 mmol/day

Page 95 of 120
 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Experiment No. 16
CREATININE DETERMINATION

OBJECTIVES: At the end of the activity, the students are expected to:

 Discuss the sources, metabolism, formation of Creatinine;

 Discuss the different factors that may affect the level of Creatinine in blood;
 Describe the principle involved, advantages of the commonly used methods for Creatinine
determination;
 Recognize the effect of age, gender and other factors on the value of Creatinine;
 Consistently maintain quality control in patient preparation, specimen collection, sample processing & handling;
 Appreciate the significance of understanding how metabolism influences the values of various analytes in blood.
 Perform accurately assays for Creatinine.

Reference Ranges: Male: 0.7 – 1.2 mg/dL (62 - 105μmol/L)

Female: 0.6 – 1.1 mg/dL (53 - 97μmol/L)
Conversion Factor: 88.4

Method: Modified Jaffe Reaction

Principle:
Creatinine reacts with picric acid in an alkaline environment to form a red-colored product (creatinine
picrate). The intensity of the red color is directly proportional to creatinine concentration which can be measured at
500-520 nm. The use of a surfactant and sodium tetraborate keeps interferences at minimum

Procedure:
BLANK STANDARD SAMPLE

Creatinine Working Reagent 1.0 mL 1.0 mL 1.0 mL

Incubate at 37 C for 5 minutes

water 100 μL - -

Standard reagent - 100 μL -

serum - - 100 μL

 Mix; incubate for 60 seconds at 37 C, then record absorbance as A1. After exactly 60
seconds, record again absorbance as A2.
 Read the absorbance against reagent blank at 510 nm.

Page 96 of 120
Computation of results:
Creatinine mg/dL = A2 – A1 (sample) x 2 (standard value)
A2 – A1 (standard)

Specimen / Stability: serum, plasma, urine

Creatinine is stable 24 hours at 2 – 8 C. Freeze samples for prolonged storage

Linearity: the method is linear for creatinine values up to 20 mg/dl.

Interferences:
No interference was observed by the presence of:
Hemoglobin ≤200 mg/dL
Bilirubin ≤7 mg/dL
Lipids ≤600 mg/dL

GUIDE QUESTIONS:
1. How is creatinine produced in the body?
2. Describe the renal handling of creatinine.
3. Completely describe the creatinine clearance test.
4. Why is the serum creatinine determination a better indicator for renal status than serum urea
determination?

Page 97 of 120
 URIC ACID

Biochemistry
− major product of the catabolism of purine nucleosides: adenosine & guanosine
− formed in the liver & intestinal mucosa from xanthine
− The bulk of purines ultimately excreted as uric acid in the urine arises from degradation of
endogenous nucleic acids.
− Reutilization of the major purine bases (adenine, hypoxanthine and guanine) is achieved through “salvage”
pathways
• Phosphoribosylation of the free bases causes re-synthesis of the respective nucleotide
monophosphates

RENAL HANDLING of URIC ACID

• 75% is excreted through the urine
• The remainder is secreted into the GIT, where it is degraded to allantoin & other compounds by
bacterial enzymes.
− Glomerular filtration
− Tubular reabsorption in the PCT: 98 – 100%
− Active secretion
− Reabsorption in the DCT
− Net excretion: 10%

FACTORS AFFECTING URIC ACID VALUE:

1. Diet: legumes, seeds, internal organs
2. Age & gender: increase w/ age; higher in males
3. 2x greater concentration in RBC than in plasma
4. Avoid the use of K oxalate because it forms salts that cause turbidity
5. UA is stable in serum for several days at RT and longer at ref. temp.
6. Thymol increases its stability

METHODS of DETERMINATION:

1. DIRECT METHOD: Phosphotungstic Acid (PTA)

Uric Acid + PTA ------- OH-------- Allantoin + CO2 + Tungsten blue (710 nm) Alkaline

solution: NaCN: Folin Na2CO3: Caraway

Brown Henry
Benedict Archibald
Newton

3. INDIRECT METHODS: ENZYMATIC METHODS

A. Blaunch and Koch (UV test with uricase)

Uric Acid Allantoin + CO2

• The decrease in the UA concentration is determined by measuring the absorbance in the range of 290 – 300
nm

Page 98 of 120
 B. Trinder – Uricase method
Uric Acid + O2 + 2H2O Allantoin + CO2 + H2O2

H2O2 + DHBS + PAP Quinoneimine derivative (480 – 550 nm)

DHBS: 3,5 – dichloro – 2- dihydroxy benzene sulfonic acid

PAP: 4 – aminophenazone

C. Uricase – catalase system

Uric Acid Allantoin + CO2 + H2O2

H2O2 + methanol --------------- formaldehyde + H2O

(Catalase)

Formaldehyde + acetylacetone + NH3 3H2O + 3,5-diacetyl-1,4-

dihydrolutidine (410 nm)

OR

H2O2 + ethanol --------------- Acetaldehyde + H2O

(Catalase)

Acetaldehyde + NAD --------------- Acetate + NADH (increase in Abs at 340 nm)

(Aldehyde DH)

4. METHODS based on REDUCING PROPERTY of UA

A. Bittner method

Cupric ions -----UA--- Cuprous ions

Cuprous ions + neocuproine copper neocuproine complex
(yellow to orange)
B. TPTZ Method by Morin
Ferric ions -----UA--- Ferrous ions
Ferrous ions + TPTZ blue colored complex (590 nm)
TPTZ : 2,4,6- tripyridyl – 5 – triazine

4. OTHER METHODS:
A. HPLC
B. Amperometric Principle: Polarographic method

DISEASE CORRELATIONS:

(1) HYPERURICEMIA
A. Increased Formation
Primary: - Idiopathic
- Inherited metabolic disorders
Secondary: - Excess dietary purine intake
- Increased nuclear breakdown (e.g. Leukemia)
- Psoriasis
- Altered ATP metabolism
- Tissue hypoxia
- Pre-eclampsia
- Alcohol

Page 99 of 120
B. Decreased Excretion
Primary: - Idiopathic
Secondary: - Renal failure
- Drug therapy: salicylate
- Poisons: heavy metal
- Pre-eclampsia
- Organic acids
- Trisomy 21 (Down syndrome)

Hereditary Hyperuricemia:
 Lesch-Nyhan syndrome : x-linked genetic disorder; deficiency of hypoxanthine guanine
phosphoribosyl transferase (muricase)
 Abnormal phosphoribosyl pyroPO4 synthetase: prevents reutilization of purine bases in the
nucleotide salvage pathway

GOUT: monosodium urate precipitates from supersaturated body fluids

(2) HYPOURICEMIA:
o Atrophy of the liver

Specimen Requirements and Interfering Substances

− heparinized plasma, serum or urine
− immediate separation from red cells to prevent dilution by intracellular contents
− non-fasting sample acceptable
− gross lipemia should be avoided
− high bilirubin may cause false decrease
− significant hemolysis will lower results
− drugs : salicylates and thiazides : false increase

Reference Interval (uricase method)

Adult female plasma or serum 2.6-6.0 mg/dL (0.16-0.36 mmol/L) Adult male
2.5-7.2 mg/dL (0.21-0.43 mmol/L)
urine, 24h 250-750 mg/day (1.48-4.43 mmol/day) Child
plasma or serum 2.0-5.5 mg/dL (0.12-0.33 mmol/L)

Conversion factor: 0.059

Page 100 of 120

 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Experiment No. 17
BLOOD URIC ACID DETERMINATION

OBJECTIVES: At the end of the activity, the students are expected to:

 Discuss the sources, metabolism, formation of Uric acid;

 Discuss the different factors that may affect the level of Uric acid in blood;
 Describe the principle involved, advantages of the commonly used methods for Uric acid
determination;
 Recognize the effect of age, gender and other factors on the value of Uric acid;
 Consistently maintain quality control in patient preparation, specimen collection, sample processing & handling;
 Appreciate the significance of understanding how metabolism influences the values of various analytes in blood.
 Perform accurately assays for Uric acid.

Reference Ranges: Conventional S.I. Unit

Male: 3.5 – 7.2 mg/dL 0.21 - 0.42 mmol/L
Female: 2.6 – 6.0 mg/dL 0.15 - 0.35 mmol/L

Conversion Factor: 0.059

Method: Modified Trinder-Uricase/ POD Principle:
Uric acid in the sample is oxidized by uricase to allantoin and hydrogen peroxide. Hydrogen peroxide reacts
with ADPS and 4-aminoantipyrine in the presence of peroxidase to yield quinoneimine dye. The intensity of the dye is
measured photometrically (510-560) and is directly proportional to the concentration of uric acid in the sample.

Procedure:
BLANK STANDARD SAMPLE
Reagent 1.0 mL 1.0 mL 1.0 mL
water 25 μL - -
Standard reagent - 25 μL -
Serum - - 25 μL

 Mix; incubate at 37 C for 5 minutes.

 Read absorbance of standard (As) and sample (Ax) against reagent blank .

Computation of results:

Ax (patient) X 10 mg/dL (standard value)= conc. of patient (mg/dL) As

(standard)

Specimen/Stability: Serum or heparinized plasma. Oxalate, citrate and fluoride could yield a small decrease in uric
acid. Uric acid in serum is stable for 5 days at 4 C

Page 101 of 120

Linearity: method is linear to uric acid values up to 30 mg/dl.
Interferences: No interference was observed by the presence of:
Hemoglobin ≤50 mg/dL
Bilirubin ≤33 mg/dL
Lipids ≤1,200 mg/dL

• The following substances interfere in very high doses; a-methyldopa, L-dopa, 3,4
dihydroxyphenylacetic acid, and gentisic acid.

GUIDE QUESTIONS:
1. Illustrate the breakdown of purines to yield uric acid.
2. Differentiate osteoarthritis from gouty arthritis.
3. Completely describe Lesch-Nyhan syndrome and provide its association with hyperuricemia.
4. Give the clinical significance of hyperammonemia.

Page 102 of 120

 AMMONIA

Biochemistry
− from deamination of amino acids thru the action of digestive and bacterial enzymes on proteins in the GIT
− used in the liver for urea production
− level in circulation is extremely low (15 – 45 µg/dL)
− increased in concentration in the blood in cases of severe liver damage
− most ammonia in the blood exists as ammonium ion
− concentration is not dependent on renal function
− high ammonia : neurotoxic --- encephalopathy

METHODS for AMMONIA DETERMINATION:

1. Conway and Cook Diffusion Method
• specimen is alkalinized to convert NH4 ions to NH3
• NH3 is trapped in acid medium of diffusion cell
• Quantitated by titration or colorimetry
• Time consuming with poor accuracy and precision

2. Forman’s Resin Absorption Method

• Uses cation-exchange resin

• NH3 absorbed by the resin and eluted

• Quantitated by Berthelot reaction or by Nesslerization N.V.:
16 – 33 µmol/L

3. Kunahashi, Ishihora and Euhera Method

• NH3 is obtained through the use of a Dowax column
• Assayed using the Berthelot method

4. Van Anken Enzymatic Method

2-oxoglutarate + NH4+ + NADPH Glutamate + NADP + H2O

N.V.: 11 – 35 µmol/L

5. Ion Selective Electrode

• Based on the diffusion of NH3 through a selective membrane into NH4 chloride causing pH change
which is determined potentiometrically
• Good precision and accuracy

SOURCES OF AMMONIA CONTAMINATION:

1. Smoking
2. Laboratory atmosphere
 Blood collection & NH3 analysis must be done in a lab w/ restricted traffic
 Glassware: soaked in hypochlorite solution (52.5g/L)
3. Poor venipuncture technique
 Use of heparin lock
 Probing for a vein
 Partial fill of the evacuated tube
 Drawing blood into a syringe & transferring it into an anti-coagulated tube
4. Metabolism of nitrogenous constituents

Page 103 of 120

Minimized by:
o placing the specimen in ice water
o centrifuging w/o delay
o performing the assay immediately

CLINICAL SIGNIFICANCE

I. PRIMARY OR INHERITED HYPERAMMONEMIA:

A. Enzyme defects in the Kreb’s Henseleit Cycle
B. Defects in the metabolism of amino acids: Lysine & Ornithine
C. Defects in the metabolism of: Propionic acid
Methylmalonic acid
Isovaleric acid
II. ACQUIRED HYPERAMMONEMIA
A. Severe liver disease:
o Acute – toxic or fulminant viral hepatitis & Reye’s syndrome
o Chronic – cirrhosis

Hepatic encephalopathy among cirrhotic patients is caused by:

• GIT bleeding
• Excess dietary proteins
• Constipation
• Infections
• Drug effects

B. Impaired venous drainage (from intestine to liver by portal vein)

C. Impaired renal excretion Decreased urine output Increased BUN
reabsorbed increased excretion of urea into intestines converted to ammonia

Reference Interval

adult plasma 19-60 ug/dL 11-35 umol/L

urine,24h 140-1500 mg N/day 10-107 mmol N/day
child 10days to 2y 68-136 ug/dL 40-80 umol/

Page 104 of 120

 CHAPTER IX: LIPIDS AND LIPOPROTEINS

Desired learning outcomes:

At the end of this chapter, you should be able to:

1. Define lipids and lipoproteins.

2. Discuss the major classes of lipoproteins
3. Estimate plasma lipid thru different techniques
4. Discuss the different diseases related to lipoproteins.

Lipids : organic substances which are soluble in nonpolar organic solvents (chloroform and ether) and insoluble in polar
solvents (water)
✓ chemically : yield fatty acids on hydrolysis OR complex alcohols the can combine with fatty acids to form
esters
✓ human plasma lipids : cholesterol, triglycerides, phospholipids and non-esterified fatty acids
✓ Lipids are diverse in terms of their structure and function

Lipoproteins: macromolecule which consists of varying proportions of protein, cholesterol, triglyceride and
phospholipid
✓ water-soluble, thus, it facilitates the transport of the lipids in the circulation

TRIGLYCERIDE / Neutral FAT

 Secondary energy source

 Serves as a thermal insulator
 Protects tissues from physical trauma

CHOLESTEROL

 Precursor of biological hormones

 Source of bile acids
 Component of the Cell membrane

PHOSPHOLIPIDS

🞇 Component of the Cell membrane

🞇 Associated with vital life processes (e.g. CNS)

BIOCHEMISTRY OF TRIGLYCERIDES
− also known as triacylglycerol
− transported by chylomicrons (exogenous) and VLDL (endogenous)
• Complete Hydrolysis: 3 FA + glycerol Partial
Hydrolysis: 2 FA + monoglyceride
• Absorption: Glycerol (H2O-soluble) = via the portal route
Fatty acids (H2O-insoluble) = via the lymphatic route Monoglyceride
(H2O-insoluble) = via the lymphatic
route
• Synthesis of TG from monoglyceride & glycerol
• β-oxidation of fatty acids in the mitochondria: degradation of the fatty acids by 2C atoms with
subsequent production of Acetyl CoA

Page 105 of 120

BIOCHEMISTRY OF CHOLESTEROL
− found exclusively in animals and humans
− contains cyclopentanoperhydrophenanthrene ring
− contains 27 C atoms

Absorption
− cholesterol in the intestines comes from the diet, bile, intestinal secretions and cells
− to be absorbed, cholesterol has to solubilized by the formation of mixed micelles containing
unesterified cholesterol, fatty acids, monoglycerides, phospholipids and conjugated bile acids
Synthesis
− 90% of body's cholesterol is synthesized by the liver and gut from simpler molecules like Acetyl CoA

Esterification
− complexing of cholesterol with a fatty acid; this process enhances the lipid carrying capacity of
lipoproteins
− Cholesterol is mainly esterified in the vascular compartment
− Catalyzed by:
− LCAT – Lecithin Cholesterol Acyltransferase (in plasma)
− ACAT – Acyl Cholesterol Acyltransferase (intracellularly)
Transport
− transported by LDL to the cells
− transported by HDL out of the cells
Catabolism
− cholesterol that reaches the liver is either secreted unchanged into bile (free cholesterol) or
metabolized to form bile acids

LIPOPROTEINS: MAJOR CLASSES

1. Chylomicron (CM)
1. Synthesized & released from the SI (Exogenous pathway)
2. very rich in triglycerides (80%); transports dietary fat
3. relatively poor in cholesterol, phospholipids and proteins
4. Proteins: Apo B-48, AI, AII, AIV, C (1-2%)
5. when present in high levels : milky plasma (floating creamy layer)

2. Very-Low-Density Lipoprotein (VLDL)

6. synthesized and released from the liver
7. transports cholesterol & triglyceride which are synthesized in the liver (endogenous pathway)
8. smaller than chylomicrons
9. elevated levels produce a turbid plasma
10. contain : 50% triglycerides, 40% cholesterol and phospholipid, 10% protein
11. Proteins: Apo B-100 & Apo C-II

3. Intermediate Density Lipoprotein (IDL)

- derived from VLDL hydrolysis by lipoprotein lipase (LPL)
- partly depleted of triglyceride
- contains an almost equal amount of triglyceride & cholesterol
- Proteins: Apo B and E

4. Low-Density Lipoprotein (LDL)

- produced from the action of LPL on IDL

Page 106 of 120

 - consists of 50% cholesterol, 25% protein, 20% phospholipid and 5% of triglycerides

5. High-Density Lipoprotein (HDL)

- produced & catabolized in the liver & intestines
- Contains Apo AI & Apo AII
- richest in protein (50% of its weight is protein)
- involved in reverse cholesterol transport (excess cholesterol is returned from the tissues to the
liver)

ABNORMAL LIPOPROTEINS:

Lipoprotein(a) or Lp(a) – similar to LDL in terms of density and overall composition

LpX Lipoprotein – abnormal lipoprotein found in patients with obstructive biliary disease, and in patients with familial
lecithin:cholesterol acyltransferase (LCAT) deficiency

β-VLDL (“floating β” lipoprotein) – abnormal lipoprotein that accumulates in type 3

hyperlipoproteinemia
- Richer in cholesterol than VLDL

APOLIPOPROTEINS – protein portion of lipoproteins

• Apolipoprotein A : major protein of HDL and chylomicron

- originate in the intestine or liver
• Apolipoprotein B : major protein of all lipoproteins except HDL
- Two forms: Large B or B100 (found in lipoprotein formed in the liver) Small
B or B48 (found in lipoprotein formed in the S.I.)
• Apolipoprotein C : major protein of VLDL and chylomicrons and a minor protein of HDL and LDL
• Apolipoprotein D : function as a transfer protein
• Apolipoprotein E : plays a significant role in the recognition & catabolism of chylomicron remnant and IDL
via specific receptors in hepatic cells

Blood Sampling and Storage

1. Biologic variation
2. fasting – ideally fast for 12 hours3.
3. Posture- 10% decrease noted in TC, LDL-C, HDL-C after 20 mins of recumbent position4.
4. Venous vs capillary samples – capillary levels generally lower than venous
5. Plasma vs serum – either can be used when only cholesterol, triglycerides and HDL-C are
measured and LDL-C is calculated from these three measurements
6. Storage – generally, frozen samples can be satisfactorily analyzed

ESTIMATION of PLASMA LIPIDS

A. COLORIMETRIC DETERMINATION of CHOLESTEROL

Stepwise Methods
1. Direct Colorimetric / One-Step
- serum is combined with the color reagent then incubated to allow color reaction to occur Methods:
Watson’s, Ferro-Ham, Pearson, Zlatkis

a. Liebermann-Burchard Reaction
Reagents: Acid anhydride, conc. H2SO4
End-product: cholestapolyene sulfonic acid (emerald green)

Page 107 of 120

 b. Salkowski’s Reaction Reagents:
conc. HAc, Ferric ions
End-product: cholestapolyene carbonium ion (reddish-purple)
2. Two-Step Method
- Involves 1extraction step prior to 2colorimetric reaction
- Eliminates protein interference
Methods: Carr-Drekter, Saifer-Kammer, Leoffler-McDougold

3. Three-Step Method
- Requires 1extraction w/ petroleum ether followed by 2 saponification and then 3colorimetric
determination
- Standard method
- Method: Abell-Kendall

4. Four-Step Method
- Requires 1extraction, 2saponification, 3purification with digitonin then 4colorimetric
determination
- Considered as the reference method
- Methods: Schoenheimer-Sperry, Sperry-Webb

B. ENZYMATIC METHODS
− cholesteryl ester + water ----cholesterol esterase ------------ > cholesterol + FFA
− cholesterol + O2 ----cholesterol oxidase----> cholest-4-en 3-one + H2O2

DETERMINATION OF TRIGLYCERIDES
1. Colorimetric
a. Hantzsch-Lutidine Reaction (most common)
Formaldehyde + acetylacetone + NH4 3,5-diacetyl-1,4-
dihydrolutidine (yellow
@ 410 nm)
b. Van Handel and Zilversmit
Formaldehyde + H2SO4 + chromotropic acid (CTA) pink
chromophore
2. Enzymatic

a. Weiland Method
glycerophosphate + NAD ---glycerophosphate DH ---> DHAP + NADH + H NADH +
tetrazolium dye ---diaphorase---> formazan + NAD (340nm)

b. Trinder Reaction
glycerophosphate + O2 ---glycerophosphate oxidase---> DHAP + H2O2 H202 +
reduced chromogen -- Quinoneimine dye

c. Eggstein and Kreutz method

ADP + PEP ---pyruvate kinase ---> ATP + pyruvate pyruvate +
NADH + H ---LDH---> lactate + NAD (340 nm)

HDL-C Determination
− automated homogenous assays
− enzymatic method

Page 108 of 120

LDL-C Determination
Friedewald calculation: for calculation of LDL-c and VLDL-c
− cannot be used when TG is >400 mg/dL

LDL-c = TC – HDL-c – plasma TG mg/dL

5
VLDL-c = plasma TG mg/dL
5
VLDL-C in mmol/L = plasma TG
2.175
DeLong, 1986:

VLDL in mmol/L = plasma TG

2.825
VLDL in mg/dL = plasma TG
6.5

Adult Treatment Panel III or ATP III Classification for LDL, Total & HDL Cholesterol and Triglyceride Values
LDL Cholesterol (mg/dL)
<100 Optimal
100 – 129 Near optimal/above optimal
130 – 159 Borderline high
160 – 189 High
≥ 190 Very high

Total cholesterol (mg/dL)

<200 Desirable
200 – 239 Borderline high
≥ 240 High

HDL cholesterol
<40 Low
≥ 60 High

Triglycerides
<150 Normal
150 – 199 Borderline high
200 – 499 High
≥ 500 Very high

Dyslipidemias
− diseases associated with abnormal lipid concentrations
− multifactorial :
− genetic abnormalities
− environmental/lifestyle imbalances
− secondary to other diseases

Page 109 of 120

 FREDRICKSON CLASSIFICATION OF HYPERLIPOPROTEINEMIA
TYPE LIPOPROTEIN PLASMA LIPID
DISORDER STANDING TEST ABNORMALITY
Type I Hyperchylomicronemia Creamy layer; Clear Mod. Increase to
to Slightly Turbid markedly increased
TG

Type IIa Increased LDL Clear w/ Yellow Increased

- Premature Orange tint cholesterol &
atherosclerosis normal TG
- Familial
Hypercholesterolemia

Type IIb Increased β and pre-β Clear to Slightly Increased cholesterol

due to deficiency of LDL Turbid & mod. Increased TG
receptors

Type III Broad merging of β Thin cream layer Increased cholesterol

and pre-β Increased Turbid to opaque & mod. to marked
IDL increase in TG

Type IV Inc. pre-β Turbid to opaque Normal to slight

Increased VLDL increase in
cholesterol & mod. to
marked increase in
TG

Type V Increased chylomicron & Thick creamy layer Mod. increase in

VLDL Turbid to opaque cholesterol & marked
increase in TG

Page 110 of 120

 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Experiment No. 18 LIPID

PROFILE

OBJECTIVES: At the end of the activity, the students are expected to:
 Rationalize the requirements regarding patient preparations; specimen collection; transport processing and
handling
 Discuss the principle involved, advantages and disadvantages of laboratory methods of lipid &
lipoproteins
 Enumerate the reference value of each lipid measured
 Correlate laboratory results with patients lipid or lipoprotein status & in the assessment of risk or coronary
heart disease (CHD)
 Discuss the significance played by cardiac proteins and enzymes in the diagnosis of heart diseases
 Consistently maintain quality control in patient preparation, specimen collection, sample processing & handling;
 Appreciate the significance of understanding how metabolism influences the values of various analytes
in blood.
 Perform accurately the tests included in Lipid Profile

I. Total Cholesterol

Expected values: Desirable 140 – 200 mg/dL

Borderline 200 – 240 mg/dL
High Risk >240 mg/dL
Conversion factor: 0.0259 (mmol/L) Method:
Cholesterol Oxidase (Trinder Method) Principle:

Cholesterol ester hydrolase hydrolyzes cholesterol esters to free cholesterol and fatty acids. Cholesterol
oxidase catalyzes the oxidation of free cholesterol to cholest-4-ene-3-one and hydrogen peroxide. The H2O2
reacts with p-chlorophenol and 4-aminoantipyrine in the presence of peroxidase to form a quinoneimine dye. The
intensity of color formed is proportional to the cholesterol concentration and can be measured photometrically
between 480 and 520 nm.
COLORIMETRIC PROCEDURE
BLANK STANDARD SAMPLE

Reagent 1.0 mL 1.0 mL 1.0 mL

water 10 μL - -

Standard reagent - 10 μL -

Serum - - 10 μL

🞇 Mix; incubate at 37 C for 5 minutes.

🞇 Read absorbance of standard (As) and sample (Ax) against reagent blank.

Computation of results:

Ax x 200 = Cholesterol in mg/dL

As

Specimen / Stability: serum, plasma (EDTA); sample is stable for 3 days at 2 - 8 C and 1 month at - 20 C
Linearity: method is linear for cholesterol values up to 700 mg/dL.
Interferences:

Page 111 of 120

 No interference was observed by the presence of:
Hemoglobin ≤500 mg/dL
Bilirubin ≤15 mg/dL
Lipids ≤850 mg/dL

II. HDL – C DETERMINATION:

Method: Immunologic method

Expected values: Adult male: 35.3 – 79.5 mg/dL
Adult female: 42.0 – 88.0 mg/dL

Principle:
Anti-human 3 lipoprotein antibody in Reagent A binds to lipoproteins (LDL, VLDL and chylomicrons)
other than HDL. The antigen-antibody complexes formed block enzyme reactions when Reagent B is added.
Cholesterol esterase and cholesterol oxidase in Reagent B react only with HDL-C. Hydrogen peroxide produced
by the enzyme reactions with HDL-C yields a blue color complex upon oxidative condensation of F-DAOS [N-
ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5- dimethoxy-4-fluoroaniline, sodium salt] and 4-aminoantipyrine in the
presence of peroxidase. By measuring the absorbance of the blue color complex produced, at the optimum
wavelength of 593
nm, the HDL-C concentration in the sample can be calculated when compared with the absorbance of the HDL-C
calibrator.

Test Procedure:
Mix; incubate for 5 minutes at 37 C. Read absorbance of calibrator (As) and sample (Ax)
against reagent blank.

Computation of results:

Ax x calibrator value = HDL-C in mg/dL

As

III. TRIGLYCERIDE DETERMINATION

Expected value: Desirable: <200 mg/dL (2.26 mmol/L)

Conversion factor: 0.011

Method: Trinder method

Principle:
Triglycerides are hydrolyzed by lipoprotein lipase to produce glycerol and free fatty acids. The glycerol
participates in a series of coupled enzymatic reactions in which glycerol kinase / glycerol phosphate oxidase
are involved and H2O2 is generated. H2O2 reacts with TOPS and 4- aminoantipyrine in the presence of
peroxidase to form a quinoneimine dye. The intensity of color formed is proportional to the triglyceride
concentration and can be measured photometrically at 546 nm

BLANK STANDARD SAMPLE

Reagent A 360 μL 360 μL 360 μL
water 4 μL - -
calibrator - 4 μL -
serum - - 4 μL
Mix; incubate at 37 C for 5 minutes
Reagent B 120 μL 120 μL 120 μL

Page 112 of 120

 COLORIMETRIC PROCEDURE
BLANK STANDARD SAMPLE

Triglyceride Reagent 1.0 mL 1.0 mL 1.0 mL

water 10 μL - -

standard - 10 μL -

serum - - 10 μL

🞇 Mix; incubate at 37 C for 5 minutes.

🞇 Read absorbance of standard (As) and sample (Ax) against reagent blank.

Computation of results:
Ax X 200 mg/dL As
Specimen / Stability: Fasting specimen obtained (10 – 14 hours); either serum or EDTA plasma can be used to determine
triglycerides. When EDTA plasma is used, the plasma value is converted to the equivalent serum value by multiplying the plasma
value by 1.03. Specimens are stable at 4 C for 3 days, frozen at -20 C for two weeks, or frozen at -70 C for longer periods.
Lipemic specimens may require warming at 37 C and vigorous mixing before analysis, especially if they have been frozen
Linearity: The method is linear for triglyceride values up to 1000 mg/dl

Interferences:
No interference was observed by the presence of:
Hemoglobin ≤150 mg/dL Bilirubin
≤18 mg/dL

IV. LDL-C DETERMINATION

Expected Values: 76 – 218 mg/dL

NCEP ATP III Decision cut-off points for LDL-C:

Desirable <130 mg/dL

Borderline for CHD 130 – 159 mg/dL
High Risk for CHD ≥160 mg/dL

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Method: Modified Trinder Principle:
When a sample is mixed with Reagent 1, the protecting reagent binds to LDL and protects it from enzyme
reactions. Cholesterol esterase (ChE) and cholesterol oxidase (ChO) react with non-LDL lipoproteins
(chylomicrons, VLDL and HDL). Hydrogen peroxide produced by the enzyme reactions with non-LDL cholesterol is
decomposed by catalase in Reagent 1. When Reagent 2 is added, the protecting agent is removed from LDL and
catalase is inactivated. In this second process, ChE and ChO react only with LDL-C. Hydrogen peroxide produce by
the enzyme reactions with LDL-C yields a color complex upon oxidase condensation with N-(2-hydroxy-3-
sulfopropyl)-3,5-dimethoxyaniline (HDAOS) AND 4- aminoantipyrine (4AA) in the presence of peroxidase (POD).
By measuring the absorbance of the blue color complex produced at approximately 600 nm, the LDL-C
concentration in the sample can be calculated when compared with the absorbance of the LDL-C calibrator.

Test Procedure:
BLANK STANDARD SAMPLE
Reagent A 360 μL 360 μL 360 μL
water 4 μL - -
calibrator - 4 μL -
serum - - 4 μL
Mix; incubate at 37 C for 5 minutes
Reagent B 120 μL 120 μL 120 μL

Mix; incubate for 5 minutes at 37 C. Read absorbance of calibrator (As) and sample (Ax) against reagent blank.

Calculation: Ax x calibrator value = LCL-C mg/dL

As
Specimen: serum, heparinized or EDTA plasma

Linearity: The method is linear up to 400 mg/dL

Interferences:
No interference was observed by the presence of: Hemoglobin
≤500 mg/dL
Bilirubin (free) ≤50 mg/dL
Bilirubin (conjugated) ≤40 mg/dL
Ascorbic acid ≤50 mg/dL

GUIDE QUESTIONS:
1. In tabulated form, differentiate the major lipoproteins.
2. Describe the minor lipoproteins and indicate their clinical significance.
3. Differentiate atherosclerosis from arteriosclerosis.
4. Describe the different types of hyperlipoproteinemia.
5. Describe the different types of hypolipoproteinemia.
Draw a schematic diagram illustrating lipoprotein metabolism (showing the two pathways: endogenous and exogenous

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 CHAPTER X: LIVER FUNCTION TEST

Desired learning outcomes:

At the end of this chapter, you should be able to:

A. Know the anatomy, physiology, and functions of the liver.

B. Identify the clinical manifestations of liver disease.
C. Discuss bilirubin metabolism.
D. Review the different methods used in bilirubin determination
E. Be able to identify the clinical significance of bilirubin.

REVIEW OF THE ANATOMY & PHYSIOLOGY OF THE LIVER

⚫ Largest organ (~1.2 – 1.5 kg in adult)
⚫ Located in the right upper quadrant of the abdomen
⚫ Dual Blood Supply:
a) Portal Vein – carries blood from alimentary tract
b) Hepatic Artery – carries oxygenated blood to the liver

Functions of the Liver:

1. Receives, processes & stores materials absorbed from the digestive tract
2. Synthesis: multiple plasma proteins
3. Main organ of detoxification
4. Synthesis of bile acids from cholesterol
5. Major site of catabolism of hormones

CLINICAL MANIFESTATIONS OF LIVER DISEASE

1. JAUNDICE – “Icterus”
– yellow discoloration of the plasma, skin, sclerae & mucous membranes caused by Bilirubin accumulation

2. PORTAL HYPERTENSION (>20 mmHg)

⚫ Increased: sinusoidal infiltration, scarring or hepatic vein obstruction

⚫ Major Complications: Hepatosplenomegaly, Ascites & Hepatorenal syndrome

3. HEPATIC FAILURE & ENCEPHALOPATHY

⚫ Accumulation of substances that are normally metabolized by the liver

4. ALTERED DRUG METABOLISM

⚫ Liver plays a major role in enzymatic transformation & disposition of drugs

5. ENDOCRINE ABNORMALITIES
⚫ Hormone imbalance

6. NUTRITIONAL & METABOLIC ABNORMALITIES

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7. IMMUNOGLOBULIN ABNORMALITIES
⚫ The liver efficiently sequesters dimeric IgA & secretes it into the bile

8. DISORDERED HEMOSTASIS
⚫ Chronic liver disease produces alterations in Hemostasis and a generalized hemorrhagic tendency

BILIRUBIN METABOLISM

DAILY BILIRUBIN PRODUCTION (average 250 – 300 mg)

⚫ 85%: Derived from degradation heme from hemoglobin
⚫ 15%:
FORMS OF BILIRUBIN
⚫ UNCONJUGATED – bound by albumin & transported to the liver
⚫ CONJUGATED – formed from the reaction of UCB with glucuronic acid
⚫ DELTA – conjugated bilirubin bound to Albumin

Fig. 1 Bilirubin Metabolism (Source: eclinpath.com)

METHODS OF DETERMINATION:

⚫ Jendrassik-Grof & Evelyn-Malloy procedures:

- Both have acceptable precision
- Adapted by many automated instruments

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 - Most frequently used methods to measure Bilirubin

CLINICAL SIGNIFICANCE

1) PRE-HEPATIC JAUNDICE
⚫ Occurs when the problem causing the jaundice occurs prior to liver metabolism
⚫ Caused by an increased amount of bilirubin being presented to the liver (e.g. Acute & Chronic Hemolytic
Anemia)
⚫ Unconjugated hyperbilirubinemia
2) HEPATIC JAUNDICE
⚫ Primary problem causing the jaundice resides in the liver (intrinsic liver disease)

CAUSES:

A. Disorders of Bilirubin Metabolism

A.1 Gilbert’s Syndrome

✓ intermittent hyperbilirubinemia; cause by reduction in the expression of UGT1A1 gene (thereby reduced
conjugating ability of the liver)

A.2 Crigler-Najjar Syndrome

✓ Chronic unconjugated hyperbilirubinemia; rare & more severe disorder
✓ Molecular defect w/in the gene involved with bilirubin conjugation

 Type I – complete absence of enzymatic bilirubin conjugation

 Type II – mutation causing a severe deficiency of the enzyme for conjugation

B. Disorders related to Transport (Transport Defects)

B.1 Dubin-Johnson Syndrome

✓ Removal of conjugated bilirubin from the liver cell & the excretion into the bile are defective
✓ Caused by deficiency of the canalicular transporter protein (MDR2/cMOAT)
✓ Appearance of dark-stained granules on a liver biopsy

B.2 Rotor Syndrome

✓ Hypothesized to be due to a reduction in the concentration or activity of intracellular binding proteins (e.g.
Ligandin)
✓ Liver biopsy does NOT show dark pigmented granules

3) PHYSIOLOGIC JAUNDICE of the NEWBORN

⚫ Unconjugated hyperbilirubinemia
⚫ Deficiency in the enzyme Glucuronyl transferase (one of the last liver functions to be activated in pre-natal
life)
⚫ Kernicterus – deposition of bilirubin in the nuclei of brain & nerve cells
- results in cell damage & death in the newborn

4) POST-HEPATIC JAUNDICE
⚫ Usually results from biliary obstructive disease (e.g. gallstones, tumors)
⚫ Prevents the flow of conjugated bilirubin into the bile canaliculi
⚫ Clay-colored stool

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 NAME: RATING:

Laboratory Schedule: Group No:

Date performed: Date submitted:

Experiment No. 19
DIRECT AND TOTAL SERUM BILIRUBIN DETERMINATION
(Modified Jendrassik-Grof Method)

MATERIALS AND EQUIPMENT:

1. Venipuncture set 8. Serological pipets
2. Stat fax tubes 9. Test tube racks
3. Beaker 10. Labeling tapes
4. Stat fax tubes 11. Parafilm
5. Pasteur pipets 12. Total & Direct Bilirubin Reagent
6. Serological pipets 13. Stat fax machine
7. Centrifuge 14. Applicator sticks

PRINCIPLE OF REACTION:
Direct Bilirubin: Conjugated (direct) bilirubin reacts with diazotized 2,4-dichloroaniline in acidic solution to produce
an intensely colored red diazo compound (520-560 nm). The intensity of color of this dye in solution is proportional
to the concentration of direct bilirubin.

Total Bilirubin: Bilirubin reacts with diazotized 3.5-dichloroaniline to produce an intensely colored diazo compound
(490-520 nm). The intensity of color of this dye in solution is proportional to the concentration of total bilirubin. Free
bilirubin is not soluble in aqueous media, but this reagent contains an association of surfactant and accelerators in
order to provide an accurate measurement of total bilirubin.

Reagent Composition:
Direct Bilirubin
Reagent A: 0.26M sodium chloride, 0.1Mm EDTA
Reagent B: 0.1mM EDTA, diazotized 2.4-dichloraniline 0.1mM, 0.18M hydrochloric acid
Total Bilirubin
Reagent A : 0.1M hydrochloric acid, surfactant
Reagent B: 0.1M hydrochloric acid, 3.5-dichlorophenyl diazonium salt 2mM, non-reactive stabilizers
PROCEDURE:
Direct Bilirubin
BLANK STANDARD SAMPLE
Reagent A 1.0 mL 1.0 mL 1.0 mL
water 50 μL - -
calibrator - 50 μL -
serum - - 50 μL
Mix; incubate at 25, 30 or 37 C for 5 minutes. Read absorbances of calibrator (Ac1)
and sample (Ax1) against reagent blank.
Reagent B 250 μL 250 μL 250 μL
Mix; incubate at 25, 30 or 35 C for 5 minutes. Read absorbances of calibrator (Ac2) and sample
(Ax2) against reagent blank.

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Specimen:
Fresh, hemolysis-free serum or heparinized plasma may be used. Carefully protect the
sample from light until use. Bilirubin in sample is stable for 3 days when stored in the dark
at 2-8 ˚C and 1 month at -20 C

CALCULATION:
Bilirubin (mg/dL) = Ax2 –
Ax
1 x calibrator value Ac2 – Ac1

For Factored procedure: Direct Bilirubin (mg/dL) = (Ax2 – Ax1) x 63.2

Total Bilirubin (mg/dL) = (Ax2 – Ax1) x 31.3

**To convert mg/dL to umol/L, multiply by factor 17.1.

EXPECTED VALUES:
Total Bilirubin: Adults 0.2 – 1.0 mg/dL (3.4 – 17.1 μmol/L)
Newborns:
Up to 24 hrs. 2.0 – 6.0 mg/dL (3.4 – 103 μmol/L)
Up to 48 hrs. 6.0 – 10.0 mg/dL (103 – 171 μmol/L)
3 – 5 days 4.0 – 8.0 mg/dL (68 – 137 μmol/L)

Direct bilirubin: Adults ≤0.20 mg/dL (≤3.4 μmol/L)

Linearity: Direct Bilirubin = the method is

linear up to 13 mg/dL Total
Bilirubin = the method is linear
up to 20 mg/dL

Interferences:
Direct Bilirubin: No interference was observed by the presence of:
Hemoglobin ≤50 mg/dL
Lipids ≤500 mg/dL
Ascorbic acid ≤35 mg/dL

Total Bilirubin: No interference was observed by the presence of:

Hemoglobin ≤150 mg/dL Lipids interfere

Questions for research:

1. What is the Van den Bergh reaction?
2. Aside from the method performed, describe current methods for bilirubin
determination in routine laboratories.
3. Give the reasons why the Jendrassik-Grof method is preferred than the Evelyn-Malloy method.
4. Enumerate other sources of interferences or errors that affect the bilirubin assay.
5. Describe bilirubin metabolism.
6. In a tabular form, differentiate conjugated from the unconjugated type of bilirubin.
7. What is the clinical significance of abnormal bilirubin values? Correlate with
pre-hepatic, hepatic and post-hepatic jaundice.

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