CLINICAL CONCEPTS 1

A.) BIOHAZARDS/ UNIVERSAL PRECAUTIONS

Occupational Safety & Health Administration (OSHA)
- Established by International Congress in 1970
- Authorized to create regulations, and conduct on-site inspections for mandatory safety
standards
I. Precautionary barriers – personal protective equipment (PPE)
- gloves, gowns, goggles, masks, aprons
II. Safety Practices:
a) spills
b) hoods = fume; biological
c) hand washing
d) housekeeping
e) waste disposal
f) employee training
g) labels & signs
I. Biological Safety
- Hep B, Hep C, & HIV transmission has raised concerns
- Centers for Disease Control updated its 1983 “Guidelines for Isolation Precautions in
Hospitals” by Garner
- Minimizes occupational exposures
- Reasonable anticipated skin, eye, mucous membrane, or parenteral contact with
blood or other potentially infectious materials that may result from the
performance of an employee’s duties
- All specimens – treated a potentially infectious, regardless of their status
- Blood – includes blood slides
- Body fluids – pericardial, pleural, peritoneal, synovial, amniotic, cerebrospinal
- Body secretions – semen, vaginal secretions, sputum, breast milk
- Tissues – any unfixed tissues, organs, aspiration slides
II. Fire Safety
1.) Class A Fire – involves ordinary combustible materials, such as paper, wood, plastic, &
fabric
I. Extinguisher: pressurized water or dry chemical
i. Directed at the base of flame; dangerous to use water in burning liquids
2.) Class B Fire – involves flammable liquids or gases, & combustible petroleum products
I. Extinguisher: dry chemical or CO2
i. Directed close to flame; edge, forward, & upward
3.) Class C Fire – involves energized electrical circuits regardless of fuel
I. Extinguisher: dry chemical or CO2
i. Dangerous to use water
4.) Class D Fire – involves combustible or reactive metals [Mg, Na+, K+]
I. Extinguisher: special dry chemicals
i. Trained fire-fighting personnel
III. Chemical Safety
- Federal Hazard Communication Standard require Health Care Facilities the following:
1. Employers determine if hazardous chemicals are present
2. Make Material Safety Data Sheets (MSDs) available to all employees
3. Label chemical containers with a hazard
4. Maintain a chemical inventory & hazardous chemical list
5. Provide employee information & training with PPE
 6. Develop a written hazard communication program
Information Contained in MSDs
1. Identification of Chemical (e.g., CAS no. synonyms)
2. Hazardous Ingredients/ Identity
3. Physical Data & Chemical Characteristics
4. Fire & Explosion data
5. Health Hazard Information
6. Reactivity Data (Physical Hazards)
7. Spill, Leak, and Disposal Procedures
8. Personal Protection Information (Control Measures)
9. Special Precautions and Comments
Standard Hazard Identification/ Chemical Hazard Label
- By National Fire Protection Association (NFPA)
- Utilizes a diamond color-coded symbol
- Each quadrant is graded 0-4, as to the magnitude or severity of the hazard:
- 4 – extremely hazardous
- 3 – severely hazardous
- 2 – moderately hazardous
- 1 – slightly hazardous
- 0 – none according to present data
BLUE – refers to Health Hazards; means toxic when inhaled, ingested, or absorbed through
skin; store in secure area
RED – refers to Flammable Hazards; stored in area segregated for flammable reagents
YELLOW – refers to Reactivity or Stability Hazards; oxidizing; react violently with air, water, or
other substances; store away from flammables
WHITE – refers to special Hazards; example is corrosive material; store away from colored
materials
GRAY – material presents no more than moderate hazards; store in general chemical storage

Categorie of Chemicals
1. Corrosives – chemicals with a pH of less than or equal to 2 or more than or equal to 12.5
(inhalation & contact)
a. Concentrated HCL, Acetic acid, NaOH
b. To transport this reagent one should employ a rubber carrier with handles
2. Toxic Substances – poisons, irritants, and asphyxiants (do not act directly on human
tissues but interfere with metabolic processes; enter by ingestion, inhalation, or skin
absorption)
a. Threshold limit values (TLV) found on MSDs
i. Time-weighted average (TLV-TWA) – represents the maximum allowable
exposure over an 8-hour work day
ii. Short-term exposure limit (TLV-STEL) – represents the maximum amount
of allowable exposure for a short period such as 15 minutes
iii. Ceiling value form (TLV-C) – represents the concentration of an agent
that must never be exceeded
3. Carcinogens – capable of causing cancers
a. Vinyl chloride & benzene
4. Mutagens & Teratogens – capable of causing chromosomal aberrations or congenital
malformations
5. Ignitable – flammable & combustibles; limited in 1 pint quantities if stored in glass
containers; others in safety cabinets; never store in refrigerators
6. Reactive – explosive and oxidizers
 a. With high O2 content or compounds with redox groups (hydrazine,
hydroxylamine)
b. Reacts violently with water or moist-air (anhydrous metal oxides)
c. Pyrophoric compounds that react spontaneously with air (ignites spontaneously
in air)
d. Compounds that can form peroxides over time and become explosive (diethyl
ether)
IV. Compressed Gases
- Can become torpedoes if main stem sheared by falling over
- Secure by a chain
- Use one cylinder at a time
- Use smallest size as possible
V. Electrical Safety
- Burns, shock, electrocution, ignition, and explosion
- Prevent the occurrence of high temperature
- Circuit breakers, fuses, ground fault interrupters (GFI)
VI. Radiation Safety
- Large alpha particles found in Plutonium; may cause serious tissue damage (by DNA
damage) if ingested or inhaled

B.) LABORATORY SUPPLIES & CHEMICAL REAGENTS

Validity of Clinical Laboratory Data dependent on many variables:
- Proper manipulation of equipment
- Use of specific reagents (including H2O)
- Quality of materials
- Environmental control
B.1 Laboratory Reagents
1.) Grades
a. Chemicals exists in varying degrees of purity:
b. Content actual analysis – often provided on the label
A. Reagent grades that meet specifications of American Chemical Society (ACS)/ National
Bureau of Standards:
1.) Analytical reagent-grade/ reagent-grade chemicals (more expensive but essential for
accuracy)
2.) Ultra Pure reagent – higher degree of purity
3.) Standard, Clinical Type – highest grade, most pure
4.) Spectograde; nanograde; HPLC grade (High Purity Liquid Chromatography); ACS
(American Chemical Society)
B. Reagent grades of insufficient purity:
1.) USP & NF grades
a. Meet specifications of US Pharmacopeia or National Formulary respectively
b. Of interest to pharmaceutical chemist but not of sufficient purity for chemical
analysis
2.) CP (Chemically Pure)
3.) Other designations:
a. Purified; practical; technical (lowest quality); commercial
IUPAC – specified standards: The International Union of Pure and Applied Chemistry grades
chemicals as follows:
 1.) Grade A or atomic weight standards
2.) Grade B or ultimate standards
3.) Grade C or primary standards (commercially available and have less than 0.002%
impurities)
4.) Grade D or working standards (commercially available and have less than 0.5%
impurities)
5.) Grade E or secondary standard (defined as standardized using Grade C as reference)
Buffer – a substance that resist changes in the pH of a system
Techniques of Use and Storage – read the label for proper storage
a) Most are stable at room temperature
b) Some must be refrigerated, frozen, or even stored at -70 degrees Celsius
c) Light sensitive chemicals & reagents must be stored in brown bottles
Advances in Technology – prepackaged reagents: less intensive labor
Laboratory Supplies – used for 3 Primary Purposes
- Storage, measurement, and confinement
Glass Containers
- Preferred because of chemical stability and clarity especially in chromatographic
analysis
Disadvantages of glass container
- A pH greater than 6.0 can attack glassware including borosilicate and aluminosilicate
glass
- Glass also tends to absorb metal ions and can potentially alter the concentration of
standard solution
Alkaline solutions – stored in plastic container
Glasswares
- Many grades available
- Should consist of high tensile strength, heat or light resistance
- Preffered glasswares are:
o Borosilicate
 Low alkaline-earth content (e.g. Mg, Ca,etc)
 Free of many contaminants (heavy metals)
 Can be heated to 600 degrees Celsius & will not soften until 820 degrees
Celsius (e.g. Klimax & Pyrex)
o Aluminosilicate Glass
 All the advantage of borosilicate glass
 Much greater strength
 More resistant to ordinary breakage, scratching, chipping, and clouding
(Corex & Corning)
o Soda-lime glass or standard flint glass
 Composed of mixture of oxides (silicon, Ca++, Na++)
 Poor resistance to high temperatures, and sudden changes of
temperature
 Fair resistance to attack of chemicals
 May be used only for procedures not needing to be heated or centrifuged
 Minerals may leach from and interfere
Plasticwares
- Unbreakable; may be disposable; cheaper, does not release ions
- Most are constructed from polymerized organic monomers such as:
o Polyolefin (e.g. polyethylene & polypropylene)
 o Polytetrafluoroethylene (e.g. Teflon or fluorinated hydrocarbon)
o Polystyrene (usually disposable)
o Polycarbonate & polyvinylchloride (PVC)
- Autoclavable
o Polypropylene, polyallomere, fluorinated ethylene propylene (Teflon FEP),
tetrafluoroethylene (Teflon TFE), ethylene tetrafluoroethylene (ETFE),
polycarbonate & polysulfone (PSF)
- Non-autoclavable
o Low-density polyethylene (LDPE), high density polyethylene (HDPE), and PVC
bottles
National Institute of Standards and Technology (NIST), known as National Bureau of Standards
(NBS)
- Has established standard tolerance for different volumetric glasswares used in clinical
chemistry lab
I. Volumetric Flasks
- Used in diluting a sample or solution to a certain volume (1mL to 4L)
Class A
- Flasks certified by NIST
- Designated to contain an accurate volume at a specified temperature (20-25 degrees
Celsius)
Diluting a non-acid solution
- Solution or sample is added first
- Then the diluents is added stepwise
Diluting an acid solution
- Diluent is added first
- Then the acid solution or sample is added next
- Otherwise, excess heat is generated when the diluents is added to acid
For Class A volumetric flasks, “accuracy” is used interchangeably with “tolerance” and is
expressed as ±____%
To convert or express tolerance in terms of milliliters, multiply the total capacity volume of the
flask with the % tolerance
II. Graduated Cylinders
- Made of polyethylene as well as from glass
- 10ml to 1L
- Used to measure volume of liquid when a high degree of accuracy is not essential
III. Erlenmeyer flasks
- For general mixing and preparing reagents
- 10ml to several liters
IV. Beakers
- Wide-mouth cylindrical vessels
- 5mL to several liters
- Used for general mixing and reagent preparation
CLINICAL CONCEPTS 2
Chemistry Basic Equipments
Microscope – in a microscope with a fixed tube length the magnification I obtained by
multiplying the objective and ocular magnification
I. Weighing Balances
- Used in making solutions or reagents
- Weighing done by comparing an unknown with a known mass
- Used in clinical lab for weighing small quantities
o Weight = mass x gravity
o For same gravitational force, weights should be equivalent to the masses
o Hence, in actual practice: mass ≅ weight (used interchangeably)

Types of Balance
A.) Mechanical
1.) Torsion balance
a. Found in pharmacy
b. Uses metal bands to support weight of the beam instead of the knife-edge
fulcrum
2.) Double-pan balance
a. Two weighing pans dangling from ends of the beam; knife-edge fulcrum at center
b. Substance to be weighed placed on left pan, while standard weights on the right
3.) Single-Pan balance
a. With arms of unequal length
b. Knife-edge fulcrum located near the weighing pan;
c. Arm with 2-3 parallel beam to which different size weights are attached
4.) Precision balance
a. Capable of weighing 1mg
5.) Microbalance
a. Capable of weighing 0.1µg (1mg = 1000µg)
6.) Mechanical analytical balance
a. Also called a “substitution balance”
b. Single-pan balance, enclosed by sliding transparent doors, which minimize the
environmental influences on pan movement
c. Substance to be measured placed within a “tared” (a deduction from gross
weight made to allow for the weight of paper of container) weighingvessel on the
sample pan
d. Capable of weighing 0.01mg
B.) Electronic Balances
1.) Electronic analytical balance
a. Single pan balance on arm of movable hanger
b. Utilizes magnetic force restoration cells instead of weights
c. Position of hanger is monitored by an electrical position-scanning device
d. A microprocessor converts the “compensation current” (needed to restore the
pan’s original position) into corresponding weight value in a “digital display”
2.) Electronic Single-Pan Top-Loading balance
a. Most often self-balancing
b. Coupled to a computer or recording device
c. The beam tilts downward on weighing
d. A ‘null detector’ senses the deviation of the beam from the equilibrium point
C.) Centrifuges
 - Used to separate fractions of a mixture by spinning at high speed in a circular motion
(centrifugal force)
- Used to separate serum or plasma from red cells
- Used to concentrate urine sediment
o Ultracentrifuge – used to separate lipid components such as chylomicrons from
other components
o 3 components: rotor, drive shaft, motor
o RPM – revolution per minute

Relative Centrifugal Force (RCF)

- Force required to separate 2 phases in a centrifuge
- Is the product of radial acceleration and mass of the mixture
- RCF = 1.12 x 10^-5 x r x rpm^2
- *r = radius in cm from center of rotation to bottom of tube *reported as RCF x g(gravity)
- Centrifugal analyzers utilize centrifugal force for the purpose of mixing sample and
reagents
3 Types of Centrifuges
- Swinging buckler or horizontal head
o A) centrifugation process
 Tubes at horizontal position
 Particles evenly distributed of the tube
o B) at rest
 Tubes at vertical position
 Sediments flat with supernatant above it
- Fixed angle or angle head
o Holds the tubes at specified angled (25-50)
o Particles move along side during centrifugation
o Forms sediment packed against side and bottom
o Produce less heat and friction than swinging bucket
- Ultracentrifuges
o High speed centrifuges
o Used to separate lipoproteins from serum or plasma
- Manually operated centrifuges
o Maintenance free
o Independent of external power supply
CLINICAL CONCEPTS 3
D. Pipets/Pipettes
- Used to transfer or measure aliquots of a liquid
- Clinical laboratorian should be aware when selecting type of pipet to use
- “mouth pipeting” should not be done
- Acessories:
o Pipet aid
o Pipet bulb
o Pipet tips

According to the NBS, Class A pipets are more accurate than Class B pipets. In the clinical
chemistry laboratory, the volumetric glassware must all be Class A.
Pipets may be classified as to their design (To Contain or To Deliver), drainage characteristics
(Blow-out or Self-draining), or function (Measuring/Graduated e.g., serologic, Mohr,
bacteriologic, Ball-Kolmer-Kah, micropipette; or Transfer e.g., volumetric, Ostwald-Folin,
Pasteur pipets, automated macro- or micropipette)

To Contain Pipets.

These are also called rinse-out pipets. They contain an exact amount of liquid which must be completely
transferred for accurate measurement. Examples are Micro-Folin, dual purpose, Sahli hemoglobin, Kirk
Micro, White-Black lambda, transfer micro, measuring micro, and Lang-Levy. None of these met the
Class A specification of NBS.

- calibrated by introducing the exact wt of Hg equivalent to the aqueous volume desired

To Deliver-Blow-out Pipets.

They transfer or deliver an exact amount of the liquid and are not rinsed out. Examples are Ostwald-
Folin (recognized by its bulb), serological pipets, serological long tip, and serological large tip. These
pipets are readily identified by the two forested or etched bands near the mouthpiece. The diameter of
the pipets is uniform and their volume graduations extend up to the delivery tip. The last blown-out
drop of the liquid is included in the delivered volume.

To Deliver-Non-Blow-out Pipets.

These pipets are filled and allowed to drain by gravity. The pipets must be held vertically and the tip
placed against the side of the accepting vessel.

Examples are as follows:

1) Volumetric pipets: These are the most accurate. They are Class A pipets and can be used for diluting
standards, calibrators or quality control materials. They hold and deliver specific volumes indicated at
the upper end of the pipet. After draining, the amount transferred is equal to the stated value.

2) Mohr pipets: These are also Class A pipets. Their graduations are made at uniform intervals but well
away from the tapered delivery tip. These pipets are calibrated to deliver-in between. The listed
accuracy is for the full volume; the smaller the volume used, the less accurate the volume delivered.
Note that these pipets are never blown out since they deliver volumes point to point.

MICROPIPETS.

They contain or deliver volumes ranging from 1 to 500 µL. they are also called lambda pipets. A lambda
is equal to a volume of 1 µL. Examples of micropipets are as follows:

1) Lang-Levy pipet: It is the original micropipette. It is filled to the constriction. Probably, it is the most
accurate for micro-volumes, but no longer commonly used today.
2) Disposable capillary micropipette: it consists of capillary tubing with a line marking a specific volume.
It is filled to the line by capillary action. The liquid is delivered by positive pressure (blow-out) through a
medicine dropper or an equivalent device. It is calibrated to contain therefore it requires rinsing.

3) Pasteur (glass) & Disposable (plastic) Transfer Pipets

- used to transfer liquids from 1 receptacle to another

- may be used to remove serum from a clot tube or plasma from anti-coagulated tube

4) Semi-automated Pipets

- single or multichanneled
- no pipetting bulb nor washing needed
- a plunger or trigger is used to aspirate the liquid into the pipet

Techniques used:

a) air-displacement techniques

- uses suction to draw up the fluid

b) positive displacement

- uses a mechanical device such as piston to displace the liquid to be drawn up.
- like the movement of the barrel in hypodermic syringe

- use plastic tips usually polypropylene (retain < film) (e.g. Eppendorf pipets)

Semi-automated Pipets

- Electronic Pipetters provide programmable application parameters:

a) pipetting mode - blowout, manual, rinsing

b) fixed-volume mode
c) dispensing mode - provides repetitive dispensing of constant volume.

Dispensers
- used to add repeated volumes of reagent or diluent to serial solutions or receptacles.
- consist of a reagent bottle to which a plunger with a valve system is attached.

Dessicants / Dessicators
- a compound used to absorb and remove water from the air or from another substance.
- primarily used in chemistry to prevent moisture absorption by chemicals or other compounds.

Deliquescent substance
- when a dessicant absorbs enough water from the atmosphere to cause it to become a liquid.

REFERENCE MATERIALS AND CONTROLS

Reference Materials (RMs)

- are materials with assigned known standard value because of their purity.

Standard Reference Materials (SRMs)

- certified reference material issued under NIST (National Institute of Standards and Technology)
trademark.
- NIST assigns values to SRMs and RMs, which in turn are used for calibrating and validating
instrumentation and methods and procedures.
- used to ensure accuracy, traceability, and comparability of measurement.
 Primary Reference Material
- also called primary standard, prepared from reagent grade chemicals, that are directly weighed or
measured to produce a standard solution with an exact known concentration.
- concentration or purity is known, usually 99.9% pure
Requirements:
a) stable
b) dried at 105o to 110oC w/o any physical or chem change
c) should have definite composition
d) should be prepared in a purity better than 99.0%
e) can be analyzed accurately
f) has relative high equivalent weight.

Secondary Reference Material

- a solution whose exact concentration cannot be determined by measuring the solute. It is compared to
a primary standard to assign a value.
- Concentration or purity is determined by comparison w/ a 1 degree standard

Calibrators
- are solutions that contain a known amount of an analyte and are used to calibrate an assay method.

Controls
- once a method has been calibrated, precision and accuracy must be monitored.
- patient-like substances, which are run alongside the patient samples to assess accuracy and precision
of lab tests.

Time
- NIST certifies the correct timer, which can be used to check stopwatches and timers used in the lab.

Temperature
- mercury thermometers discouraged

Thermistor - thermally sensitive resistor, usually used for detecting minute changes in temperature.

WATER PURIFICATION

Impurities that can cause significant errors:

a) Dissolved Inorganics - calcium, magnesium, zinc, iron, other salts & heavy metals
b) Organics - contaminants from soil & rain runoff e.g. detergents, tannins, humic acid;
c) Bacteria = byproducts of bacteria, pyrogens, endotoxins
d) Particles = colloids & suspended particles