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CLINICAL

CHEMISTRY
Julius P. Mario, RMT, MS Chem.

The purpose & function of laboratorians


through clinical pathology & laboratory
medicine are to assist clinicians in:
1.

Confirming or rejecting a diagnosis

2.

Providing guidelines in patient management

3.

Establishing a prognosis

4.

Detecting disease through case finding or screening

5.

Monitoring follow-up therapy

Clinical Chemistry
The systematic study of
biochemical processes associated
with health & disease &
the measurement of constituents in
body fluids or tissues to
facilitate diagnosis of disease.

SCOPE OF CLINICAL CHEMISTRY


Biochemistry
Computers

Instrumentation

CLINICAL CHEMISTRY

Pharmacology

Immunology

Endocrinology

Toxicology

Analytical Chemistry

It is much easier to detect a MALFUNCTION


than to elucidate or document the cause.
Malfunction of a cell maybe caused by a variety of
factors
1.
2.
3.
4.
5.
6.
7.
8.
9.

Destruction by trauma or by invasive agents


Genetic deficiency of a vital enzyme
Insufficient supply of one or more essential nutrients
Insufficient blood supply
Insufficient oxygen supply
Malignancy
Accumulation of waste products
Failure of a control system
Defect in the cellular recognition of certain signals

Point-of-care Testing

(POCT)
Also known as near-patient testing, alternate-site
testing or patient-focused testing

Used in emergency dept., operating suites, clinics,


health maintenance organization (HMO),
physicians, offices & nursing homes

Addresses acute patient needs

Instrumentation includes portable chemistry


analyzers, glucometers, BG Analyzers,
hemoglobin meters & coagulation testing

Specimens or samples
are analyzed
while
substances in them are
measured or quantitated.

Substances measured in serum fall generally


into the following categories:
1.

2.

Substances normally present with a function in the


circulation

glucose

electrolytes

TP

TAG, cholesterol

albumin

hormones

individual proteins

vitamins

Metabolites- nonfunctioning waste products in


the process of being cleared
ammonia
urea

creatinine

uric acid

bilirubin

3.

Substances released from cells as a result of


cell damage & abnormal permeability or
abnormal cellular proliferation
enzymes such as LD, ALT, AST, CK, AMS,
GGT, ALP & ACP
ferritin

4.

Drugs & toxic substances

antibodies

therapeutic drugs

substances of abuse

poisons

Quick Quiz

Tissue injury is greatly associated


with elevated levels of blood
a. electrolytes b. hormones
c. enzymes
d. albumin

Electrochemical methods are ideal for


the measurement of serum/plasma
a. glucose b. enzymes
c. proteins & amino acids d. metal
ions

Laboratory Tests

Chemical tests
Direct or Indirect
Colorimetric
Redox
Condensation
Degradative tests (Enzymatic)
One-step or Multi-step
NADH Consumption
Measurement of Activity (not
concentration)
Multi-method
Titrimetric
Electrochemical

Quick Quiz

Identify the analyte measured using the


following tests and the type of each test:
1. Jaffe reaction
2. Hantzsch test
3. GOD-PAP
4. Clark-Collip
5. Schales and Schales
6. Evelyn-Malloy

LAB REQUESTS

STAT = immediate; urgent requests;


emergency
= usually less than 1 hour TAT

PRIORITY = important for clinical


decisions
= less than 2 hour TAT

ROUTINE = non urgent; standard or


special
tests
= generally 4-6 hours

Glasswares

Plasticwares
Highly shock proof;
shatterproof

Breakable

Reusable

Ideal for acidic solutions


Ideal for alkaline
solutions
High thermal & corrosion
resistance
Relatively inert

Disposable

Good optical properties


Flexible
Low cost
For cryogenic

GLASS: 5 GENERAL TYPES


High thermal resistant glass

1.

Borosilicate glass with low alkali content


Resists heat, corrosion and thermal shock
Most common are Pyrex,

Kimax and Exax

Corex

- 6x stronger than borosilicate glass


- Alumina-silicate glass
- Better able to resist clouding due to to alkali or scratching
- Ideal for higher temperature thermometers, graduated
cylinders and centrifuge tubes.

Vycor

- recommended for use in application involving high


temperature and drastic heat shock and extreme
chemical treatment with acids and dilute alkalies
- heatable to 900oC and withstand downshock from 900oC to
ice water
- ideal for ashing and ignition techniques

2. High silica glass


- 96% silica content comparable to fused quartz in
its
thermal endurance, chemical stability and
electrical characteristics
- radiation resistant, good optical

qualities and temperature capabilities


- ideal for high precision analytical work and
can
also be used for optical reflectors and
mirrors
3. Glass with high resistance to alkalies
- boron-free glassware for strong alkali solutions
and digestion with strong alkalies
- less thermal resistant than borosilicate glass
- known as soft glass

4. Low actinic glass


- amber or red-colored to reduce the amount of light
passing through the substance within the glassware

- highly protective laboratory glassware for handling heat-

labile substances in the 300-500nm range


(bilirubin, carotene and vitamin A)
5. Standard flint glass
- soda-lime glass which is composed of a mixture of
oxides of Si, Ca and Na
- Lowest in cost and readily fabricated
- poorly resistant to high temperature and sudden changes in
temperature
- easy to melt and shape thus used as

reagent bottles

and
disposable laboratory glasswares

Quick Quiz

Which of the following is an aluminumsilicate glass that is at least six times


stronger than borosilicate and is resistant
to alkaline etching and scratching?
a. Kimax b. Pyrex
c. Corning boron free d. Corex
The tinted glass which is ideal for delayed
testing of light-sensitive substances is
a. standard flint
b. soda lime
c. low actinic d. boron-free

PLASTICWARES
1.

Polyolefins (polyethylenes,
polypropylenes)
Unique group of resins with relatively inert
chemical properties

Unaffected by acids (however, conc. sulfuric acid


slowly attacks polyethylene at RT), alkalies,
salt solutions and most aqueous solutions
Aromatic, aliphatic and chlorinated hydrocarbons
cause moderate swelling at RT
Organic acids, essential oils and halogens slowly
penetrate these plastics

Polyolefins (polyethylenes, polypropylenes)

Strong oxidizing agents attack this group at


elevated temperatures only

Polypropylene is more expensive


than
used in

polyethylene therefore the latter is


most disposable plastic wares

Polypropylene is sterilizable
however it absorbs pigment and tends to
become discolored

PLASTICWARES

2. Polycarbonate resin

twice as strong as polypropylene (from 100oC to


+160oC)

Chemical resistance is not as wide as the


polyolefins

Unsuitable for use with bases as amines,


ammonia and alkalies as well as oxidizing agents

Glass-clear and shatterproof

Ideal for centrifuge

tubes and
graduated cylinders

3. Tygon

Nontoxic, clear plastic of modified PVC


(polyvinylchloride)

Used extensively for the manufacture of ACA

tubings

Flexible

and used to handle most


chemicals

Can be steam-autoclaved or chemically


sterilized

Tubing is soft and flexible and quickly slips over


tubulatures but gripping tightly on glass or

4. Teflon fluorocarbon resins

Almost chemically inert with high corrosion

resistance at extreme temperatures

For cryogenic experiments and work at

high temperatures over extended


periods (-270oC to +255oC)

Pure translucent white and inert

Easy to clean and fast drying but it can be


scratched and misshaped

Quick Quiz

The internal tubings of clinical


analyzers should be made of
a. Teflon fluorocarbon b.
polycarbonate
c. Tygon d. polyvinylchloride

The advantages of this plastic is that it


can be made into very thin sheets and
can withstand extreme temperatures.
a. Polyolefins b. Polycarbonate
c. Tygon d. Teflon

PIPETS
Two General Types
1.

Volumetric or Transfer pipet


Designed to

liquid

deliver (TD) a fixed volume of

Consists of a cylindrical
narrower glass tubing

bulb joined at both ends to

Calibration mark is etched around the upper suction tube


Lower delivery tube is drawn out to a fine tip
used for accurate

measurements of aliquots
of nonviscous samples, filtrates, controls,
and standard solutions

OSTWALD-FOLIN PIPET

Similar to volumetric pipet but have their bulb


closer to the delivery tip

Used for measuring viscous fluids as


blood or serum

Has an etched ring near the


mouthpiece (blowout pipet)

Blown out only when the fluid has drained to


the last drop in the delivery tip

2. Graduated or Measuring Pipets

Plain, narrow tube drawn out to a tip

Graduated uniformly along its


length

Two types which are calibrated for delivery are


available

Mohr pipet
- calibrated between two marks on
the stem

Serological pipet
- has graduation marks down on
the tip and blown out to deliver the entire
volume of the pipet if etched ring or double
rings are evident near the mouth of the pipet

Serological pipet

Intended for the delivery of


predetermined
volumes

Principally used for the measurements of


reagents and are not generally
considered accurate enough for
measuring viscous samples
and
standards

Serological pipets have larger orifice than


the Mohr pipet and thus drain faster

MICROPIPETS
Sahli (TC)
Kirk (TC)
Lang-Levy (TD/ TC)
Overflow (TC)
Capillary (TC)
Sanz Pipet (TD=TC)
Unopette (TC)
Semiautomatic pipet Eppendorf (TD/TC)

Quick Quiz

The College of American Pathologists


(CAP) requires that volumetric pipets and
flasks be certified as
a. Class A b. Class B
c. Class C d. Class D

To deliver(TD) pipettes are identified by


a. two etched bands near the
mouthpiece
b. self-draining capacity
c. dual purpose pipet labels
d. blue graduation levels

CHEMICALS

The American Chemical Society (ACS) established the


specifications of chemicals in the analytical grade category.
The analytical reagent grade chemicals are of very high purity.
They are labeled Analytical Reagent (AR) or ACS which state the
actual purity of each lot. These include chemicals such as:

spectrograde,

nanograde, and

High Performance Liquid Chromatography (HPLC) grade.


The AR grade chemical are used in:
a. most analytical laboratory procedures
b. making up reagents for spectrophotometry, chromatography,
trace metal analysis, atomic absorption spectrophotometry,
fluorometry and standardization procedures.

United States Pharmacopoeia (USP) and the


National Formulary (NF) grade chemicals are not
injurious to humans and are used to produced
drugs. They are not pure enough for use in
most chemical procedures.
Less pure grade chemicals are referred to as
purified and technical. The chemically pure (CP)
or pure grade chemicals do not recommend for
reagent preparations.
Technical or commercial grade reagents are
primarily used in manufacturing and should not
be used in the clinical laboratory. The technical
grade is the lowest quality and should not be
used for analytical work.

STANDARDS

A primary standard (PS) is a chemical that is the


highest purity and can be measured directly to
produce a substance of exact known concentration.
The International Union of Pure and Applied Chemistry
(IUPAC) requires that
PS be at least 99.98% pure and that the
working standards be 99.95% pure.
Grade A - atomic weight standards
Grade B ultimate standards
Grade C* primary standards with >0.002% impurity
Grade D* working standards with >0.5% impurity
Grade E secondary standards derived from using
Grade C standards
*commercially available

The National Bureau of Standards (NBS) now


National Institute of Science and Technology
(NIST) purified materials known as Standard
Reference Materials (SRM) are used as primary
standard materials in the clinical laboratory. They
are relatively expensive and should be used as a
reference for the comparison of commercially
obtained standards and reagents.
A secondary standard is a substance of lower
purity whose concentration is determined by
comparison to a primary standard.

Quick Quiz

Regarded as the ultimate standard by IUPAC is


a. Grade A
b. Grade B
c. Grade C
d. Grade D
What is the most common use of a standard
in the clinical laboratory?
a. to verify accuracy of results
b. to standardize the testing
c. to compare its value with the results
obtained
d. to obtain the concentration of the
substance tested

WATER
Reagent grade water (RGW) is water

suitable for reagent and standard preparation. Most


procedures use distilled water or deionized water.

Distilled water is purified to remove almost all


organic materials. Water may be distilled more than
once and each distillation cycle will remove
impurities.
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.

The College of American Pathologists (CAP) and the National


Committee for Clinical Laboratory Standards (NCCLS) issued
minimum specifications for RGW. The NCCLS requirements for each
type are tabulated below:
Specifications

Type I

Bacterial content (maximum


colony forming unit/mL)
pH NA

Type II

Type III

<10

102 NA

NA NA

Resistivity (megaohms/cm 1.0 (in-line) 1.0


at 25oC)
Silicates, mg/L SiO2 maximum

0.05

0.1
0.1 1.0

Particulate matter (filter in system) 0.22 m


Organic contaminants
filter in system

activated carbon

NA
NA

NA

NA

Type I RGW is used for procedures that require maximum


water purity such as :
preparations of standard solutions,
ultramicro chemical analysis,
measurement of nanogram or subnanogram
concentration
and tissue or cell culture (or both).

Type II RGW is used in most laboratory determination in

chemistry, hematology, microbiology, immunology and other


chemical laboratory areas.

Type III RGW is used in most qualitative measurement /

examinations, most procedures in urinalysis, parasitology and


histology, washing glasswares and procedures not requiring
Type I or Type II water.

Carbon dioxide-free water is obtained by boiling Type II water. This


is used when carbon dioxide, ammonia and oxygen may affect
analysis.

CLEANING GLASSWARES AND PLASTIC


WARES

Routine washing may be done by soaking in dilute bleach followed


by drying in an oven, soaking in 20% nitric acid solution for 12-24
hours and soaking in acid-dichromate solution.

For blood clots, soak in 10% NaOH

For new pipets, soak in 5% HCI or 5% HNO3

For metal ion determination, soak in 20% nitric acid

For grease, soak in any organic solvent or 50% KOH.

For permanganate stains, soak in 50% HCI or a mixture of 1%


ferrous sulfate in 25% sulfuric acid

For bacteriologic glassware, soak in 2% to 4% cresol solution


followed by autoclaving and thorough washing

For iron determination, soak in 1:2 dilution of conc. HCI solution or


1:3 dilution of conc. HNO3

Quick Quiz

What is the most appropriate term to


describe water that is used as a solvent
in the clinical laboratory?
a. distilled water b. deionized
water
c. reagent grade water d. tap water

The type of water desired for use in test


methods requiring maximum accuracy
and precision is
a. distilled b. pure grade
c. type I
d. type II

What is effectively removed by activated


charcoal adsorption in water purification?
a. charged chemicals
b. microbes and
pyrogens
c. toxic gases
d. organic compounds

What purification method is ideal for the


removal of pyrogens and microorganisms?
a. activated charcoal adsorption
b. semipermeable membrane filtration
c. reverse osmosis
d. distillation and deionization combined

Chemical Hazards
Caustic can cause burns
Flammable also combustible, that is,
can
easily catch fire
Corrosive harmful to mucous
membranes,
skin, eyes or tissues
Carcinogen can produce or incite cancer
Mutagen cause changes in RNA or
DNA
Teratogen cause birth defects

Quick Quiz

Xylene, ethanol, methanol, and acetone


would be in which hazard class?
a. corrosive
b. flammable
c. oxidizer d. all of the above

Precautions such as using a fume hood,


wearing rubber gloves, donning a
respirator, and cleaning glassware with a
strong acid or organic solvent are
consistent with working with
a. corrosives b. carcinogens
c. azides d. all reagents

CENTRIFUGE
The relative centrifugal force (RCF) is obtained by
using a nomogram or by using this formula:

RCF = 1.118 x 10-5 x r x rpm2


Where: r = radius in cm. between the axis of rotation and the center of
the centrifuge tube
rpm = speed in revolutions per minute

The CAP recommends cleaning of any spills or debris


such as blood, glass and others daily; checking of the
timer, brushes and speed every three months. The speed
of the centrifuge is checked using the tachometer or

strobe light.

All laboratories are required to have safety


showers, eyewash stations and fire
extinguishers and to periodically test and
inspect the equipment of proper operation.
Other items that must be available for
personnel include fire blankets, spill kits and
first aid supplies.

Mechanical pipetting devices must be used


for manipulating all types of liquids in the
laboratory, including water. Mouth

pipetting is strictly
prohibited.

Quick Quiz

Which of the following may be safely


pipetted by mouth?
a. serum b. urine
c. saline solution d. none of the above

The speed of the centrifuge should be


checked at least once every three (3)
months with a /an
a. tachometer b. wiper
c. potentiometer d. ergometer

BIOLOGIC SAFETY

All samples and other body fluids should be


collected, transported, handled and
processed using strict precautions.

Gloves, gowns and face protection must be


used if splash or splattering is likely to
occur.

Specimen should remain capped during


centrifugation because biologic
specimens could produce finely
dispersed aerosols that are a high-risk
source of infection.

Any blood, body fluid or other potentially infectious material spill


must be cleaned up and the area of equipment disinfected
immediately.

Recommended clean up includes the following:


A.
B.

C.

D.
E.

F.
G.

Wear appropriate protective equipment


Use mechanical devices to pick up broken glass or
other sharp objects.
Absorb the spill with paper towels, gauze pads or tissue,
etc.
Clean the spill site using common aqueous detergent.
the spill site using approved disinfectant or 10% bleach
using appropriate contact time.
Rinse the spill site with water.
Dispose all materials in appropriate biohazard
containers.

Occupational Safety and Health Organization Administration


(OSHA) Blood Borne Pathogens standard requires written
Exposure Control Plan
Categories of exposure are the following:

Category I- daily exposure to blood and body


fluids
Category II- regular exposure to blood and
body fluids
Category III- no exposure to blood and body
fluid
Employers must offer Hepatitis B vaccine at no cost
to all personnel in Category I and II.
Biological safety cabinets should be installed in strategic places
to facilitate manipulations of infectious material. It reduces
risk of exposure of laboratory personnel and patients.

SAFETY AGAINST EXPOSURE TO TOXIC


CHEMICALS
OSHA published its Hazard Communication Standard in
1983 to minimize the incidence of chemically related
occupational illnesses and injuries in the workplaces.
OSHA requires the following:
a. Manufacturers of chemicals should evaluate the hazards of
the chemicals they produce and develop hazard
communication programs for employees exposed to
hazardous chemicals.
b. Clinical laboratories should develop and institute a
chemical hygiene plan.
c. Hospitals and laboratories are obliged to maintain an
inventory of all hazardous substances used in the
workplace.

OSHA requires that material safety data sheets (MSDS) be


provide for each chemical by manufacturers and suppliers
and must be made available on site for laboratory
personnel.
MSDS will specifically include

the chemical identity;

chemical and common name;

physical and chemical characteristics;

signs and symptoms of exposure;

routes of entry;

exposure limits;

carcinogenic potential;

safe handling procedures;

spill cleanup procedures; and

emergency first-aid.
MSDSs contain information on the nature of the chemical,
precautions if spilled and disposal recommendations.

The National Fire Protection Association (NFPA) has


developed the Hazards Identification system to
provide common, recognizable warning signs for
chemical hazards. The system consists of four colorcoded, diamond-shaped symbols arranged to form a
larger diamond shape.
(top) designates flammability
(left) indicates health hazards
(right) indicates reactivity-stability
(bottom) indicates special consideration

Contained within each color-coded diamond is a


number ranging from 0 to 4, indicating the severity of
the respective hazard (0 = none and 4 = extreme)

Special Considerations

Ox

Water Reactive

Oxidizing Agent

Radioactive

Poison

ELECTRICAL SAFETY

Lockout/tag out malfunctioning electrical or


mechanical equipment until services.
Report any small shocks, unplug and
tag equipment until serviced.

If a severely shocked person can not let go of


instrument, unplug it without touching
the person or knock person loose
with nonconductive material such as
wood.

RADIATION SAFETY

A radiation-safety policy should include environmental and


personnel protection.

All areas where radioactive materials are used or stored


must be posted with caution signs and traffic in these
areas should be restricted to essential personnel only.

Records must be maintained as to the quantity of


radioactive material on hand as well as the quantity that is
disposed. Records must be maintained for the length of
employment plus 30 years.
Radiation monitoring utilizes film badge or survey meter.
The exposure limit (maximum permissible dose
equivalents is 5000 mrem/year whole body)

The Wipe test (Leak test) involves wiping laboratory


surfaces with moistened absorbent material and the
radiation contained in each wipe is counted.

Quick
Quiz

A corrosive material was spilled onto the hand of


a laboratorian. After diluting the material under
running cold water, what should be done next?
a. consult the MSDS
b. wipe up spills with paper towels
c. dilute spills and remove it in a biohazard
bag
d. go to the emergency room

Which of the following information is found in the


MSDS?
a. Health hazard data
b. Fire & explosion
hazard data

c. First Aid measures

d. All of the above

FIRE SAFETY
Fires have been divided into four classes on
the nature of the combustible material and
requirements for extinguishments.
Class A- ordinary combustible solid materials
such as paper,
wood,
plastic and rubber
Class B- flammable liquids/ gases and
combustible petroleum products
Class C- energized electrical equipment
Class D- combustible/reactive metals such as
Mg, Na and K
Class E nuclear reaction
Class K cooking media

(Type A) Pressurized water extinguishers (foam and


multipurpose dry-chemical types) are for Class A fires.

(Type ABC) Multi-purpose dry chemical and carbon


dioxide extinguishers are used for Class B and C fires.

(Type C) Halogenated hydrocarbon extinguishers are


recommended particularly for use with computer
equipment.

(Type D) Class D fires present special problems and


extinguishments is left to trained fire fighters using
special dry-chemical extinguishers.

Personnel should know the location and type of portable


fire extinguisher near the work area and know how to use
an extinguisher before a fire occurs.

Quick Quiz

A fire extinguisher used in the event of


an electrical fire should include which
of the following classifications ?
a. type A b. type B
c. type C d. type D

In the Hazards Identification System,


what hazard does the blue diamond
positioned to the left identify ?
a. flammable b. health
c. reactivity
d. contact

SAFETY AWARENESS FOR CLINICAL


LABORATORY PERSONNEL

Health Care Organizations focus their responsibility in


protecting their employees from infection especially
against hepatitis B virus (HBV), human

immunodeficiency virus (HIV) & TB


bacilli.

The Centers for Disease Control (CDC) updated the


1983 Guidelines for Isolation Precautions in hospitals
with the release of its Universal Precautions in 1987
because of the growing concern about HIV.

The CDC recommends that blood and body fluid


precautions should be consistently used for all
patients regardless of their blood-borne infection
status.

Potentially infectious materials include:


a. body fluids (semen, vaginal
secretions, pericardial fluid, peritoneal fluid,
synovial fluid, pleural fluid, amniotic fluid,
saliva, tears, CSF, urine and breast milk)
b. unfixed tissues, organs or blood slides

Infective agents may be inactivated by


= heat sterilization (250OC for 15 minutes),
= ethylene oxide (450-500 mg/L at 55-60OC),
= 2% glutaraldehyde,
= 10% hydrogen peroxide,
= 10% formaldehyde,
= 5-25% hypochlorite,
= 10% v/v with tap water of a common household
bleach makes a very effective and economical
disinfectant inactivating HBV in 10 minutes
and HIV in 2 minutes.

DISPOSAL OF HAZARDOUS MATERIALS


There are four basic waste disposal techniques.
These include:
flushing down the drain to the sewer
system
Incineration
landfill

burial

recycling

It is permissible to flush water-soluble substances down the


drain with large quantities of water.

Strong acids and bases should be neutralized before


disposal

Foul smelling chemicals should never be disposed of down


the drain.

Other liquid wastes, including flammable solvents, must be


collected in approved containers and segregated into
compatible classes.

flammable material can be burned in specially designed


incinerators with after burners and scrubbers to remove
toxic products of combustion.

solid chemicals wastes that are unsuitable for incineration


must be buried in a landfill.

Biohazardous Wastes

Medical waste is defined as a special waste


from
health care facilities and is further defined
as solid waste that, if improperly treated or
handled may transmit infectious diseases.

it comprises of animal waste, bilk blood and blood


products, microbiologic wastes, pathologic
wastes and sharps.

The approved methods for treatment and


disposition of medical wastes are
incineration,
steam sterilization, burial, thermal
inactivation, chemical disinfection, or
encapsulation in a solid matrix.

Quick Quiz

The major job-related hazard in clinical


laboratories produces symptoms of malaise,
anorexia, nausea, vomiting, fatigue, diarrhea,
and abdominal tenderness is
a. AIDS
b. salmonellosis
c. tuberculosis
d. hepatitis
A laboratory professional was asked to send a
blood specimen from an HIV-positive patient to
a laboratory for special tests. The specimen
must be properly labeled and shipped as
a. infectious substance
b. diagnostic
specimen
c. biological product d. clinical specimen

UNITS OF MEASUREMENT

The Systeme Internationale (SI) defines seven base units as shown.


Quantity

Basic Unit

Symbol

Length
meter m
Mass
kilogram
kg
Time
second s
Electric current
ampereA
Temperature
Kelvin K
Luminous intensity Candela
Amount of substance
mole
Catalytic amount
katal

cd
mol

SI-Derived Units are obtained by combining two


or more basic units by multiplication or division.
Derived Quantity Derived Unit

Symbol

Substance concentrationmoles per cubic meter

mol/m3 or mol m-3

Conductance

Siemens

Resistance

Activity (radionuclide)

Becquerel

Volume cubic meter

m3

Bq

The National Institute of Standards and


Technology (NIST) recognizes the use of
five specific types of calibrating weights.
Class S weights for monthly calibration of
analytical balances
Class M weights - to calibrate the weights
themselves since their quality is that of
primary
standards.
Class S-1 - used for routine analytical work.
Class P weights - are allowed greater
tolerance levels than Class S-1
Class J weights - used for microanalytical
work.

The accuracy of the thermometer used to monitor the


incubation temperature of an instrument should be
verified every 6 to 12 months.
Reference thermometers must be certified by the NIST.
For the monitoring of enzymatic reactions ,
thermometers should agree within 0.1oC with the NIST
reference thermometer.
Thermometers used to check refrigerator and freezer
temperatures should agree within 1.0oC with the
reference thermometer. Thermometers should be
discarded if they differ by more than 1 oC from the
reference.
On a daily basis, the temperature of the incubation area
should be checked, and the thermometer itself should be
observed for splits in the mercury column.

It is imperative that preventive


maintenance procedures be performed
and the results recorded for all laboratory
instrumentation.
This includes maintenance of analytical
balances, refrigerators, freezers,
centrifuges, ovens, water baths, heating
blocks, thermometers, pipettors,
dilutors, automated analyzers, and all
other laboratory equipment used for
analyzing specimens.
Preventive maintenance is performed at
scheduled times such as per shift, daily,
weekly, monthly, or yearly.

Quick Quiz

SI units are designated units employed by


the International System of Units. The unit
class that encompasses the seven
fundamental quantities of measurement is
a. base
b. primary
c. derived d. elemental

Which of the folllowing is most commonly


represented by a derived unit ?
a. amount of substance
b. electric
current
c. mass
d. substance
concentration

FACTORS THAT INFLUENCE


LABORATORY TESTING

EFFECT ON LABORATORY
RESULTS

Increased Decreased
Exercise
Immediate effects:
Alanine and lactate
FFA
Long-term effects
CK, LD, AST, platelets,
testosterone,
androstenedione and LH
Fasting Most analytes
(8-12 hrs for common
bilirubin, FFA, TAG
metabolites and
12-14 hrs for lipid profile)
profile)

Glucose

Increased

Decreased

After eating
K, TAG (chylomicrons),
turbidity of serum and plasma

ALP intestinal isoenzyme,

Diet
High meat
NPNs (esp. urea, ammonia,
and urate except creatinine)
High ratio of unsaturated
to saturated fat
Purine-rich

Serum
cholesterol

Urates

High caffeine FFA, catecholamine release


Bananas, pineapple,
tomatoes and
avocados
Alcohol ingestion
Immediate
and FFA

Serotonin in blood
5HIAA in urine

Lactate, urate, ketone bodies,

Chronic abuse HDL-cholesterol, GGT, and MCV

Tobacco smoking
Acute effects

Chronic effects

Increased

Decreased

Catecholamines, cortisol
neutrophils, monocytes
and FFA

Eosinophil

CO-Hb, MCV and leukocyte


count

Prolonged tourniquet application Serum enzymes, protein,


protein-bound substances,
cholesterol, TAG, Ca, blood
cell concentration (hemoconcentration)**
Postural changes

same as ** due to hydrostatic


efflux of water

Stress

lactate, FFA and alteration


in electrolyte levels

Quick Quiz

Which of the following substances does not


require the patient a fasting blood
specimen?
a. uric acid
b. triglycerides
c. cholesterol d. phosphates

Expected to be increased in blood in


situations characterized by
hemoconcentration is
a. calcium b. potassium
c. ferritin d. creatinine

BLOOD COLLECTION
Venipuncture (Phlebotomy)

Venous blood is deoxygenated blood that


contains substances that come from

metabolic activities of different organs.


Venous blood is mostly used in analysis of blood
chemistry and immunologic studies.

Venous blood is more easily collected than arterial


blood.

Maybe obtained via one-movement or two-

movement techniques.
Sites of venipuncture includes:

Antecubital fossa veins (most preferred site)

median cubital veins and cephalic veins

Wrist veins, ankle veins, hand veins

Complications of venipuncture-classified as
immediate or delayed and local or systemic
Hematoma due to missed vein
Collapsed small veins due to excessive pull of
plunger
Syncope
Excessive bleeding
Thrombosis of vein
Infection is situ
Blood-borne infection like hepatitis B and
AIDS

Considerations

Clean the venipuncture site with 70% alcohol or


1% iodine.
Apply the tourniquet several inches above the
puncture site.
Never leave the tourniquet longer than one
minute.
The needle, as it enters the skin, is positioned at
approximately 15-degree angle to the site with the
bevel up.
Ensure prompt and adequate mixture of blood
and anticoagulant to prevent coagulation and to
prevent formation of unwanted blood clot.
Blood should not be drawn in an area with
intravenous line.

Safety Measures in Phlebotomy


(1)Observe universal (standard) safety precautions.
(2) Wash hands in warm, running water with the
chlorhexidine gluconate hand washing product
(approved by the Infection Control Committee), or if
not visibly contaminated with a commercial foaming
hand wash product before and after each patient
collection.
(3) Gloves are to be worn during all phlebotomies, and
changed between patient collections.Palpation of
phlebotomy site may be performed without gloves
providing the skin is not broken.
(4) A lab coat or gown must be worn during blood
collection procedures.

(5) Needles and hubs are single use and are


disposed of in an appropriate 'sharps' container
as one unit. NOTE: Needles are never recapped,
removed, broken, or bent after phlebotomy
procedure.
(6) Gloves are to be discarded in the appropriate
container immediately after the phlebotomy
procedure. All other items used for the
procedure must be disposed of according to
proper biohazardous waste disposal policy.
(7) Contaminated surfaces must be cleaned with
freshly prepared 10% bleach solution. All
surfaces are cleaned daily with bleach.
(8) In the case of an accidental needlestick,
immediately wash the area with an antibacterial
soap, express blood from the wound.

Factors to consider in site


selection

* Extensive scarring or healed burn areas should be


avoided
* Specimens should not be obtained from the arm
on the same side as a mastectomy.
* Avoid areas of hematoma.
* If an IV is in place, samples may be obtained
below but NEVER above the IV site.
* Do not obtain specimens from an arm having a
cannula, fistula, or vascular graft.

* Allow 10-15 minutes after a transfusion is


completed before obtaining a blood sample.

Which of the following are


allowed in phlebotomy?
____Observe

universal safety precautions.


___Handwashing before and after phlebotomy.
___Collecting blood without a lab gown.
___Recapping or removal of needles after
withdrawing the
needle.
___Reuse of gloves for as long as they were
autoclaved
beforehand.
___Weekly cleaning of all lab surfaces with
10% bleach solution.
___Outpatients are identified by making them
say their name
and date of birth.
___Doing the venipuncture twice on a patient.

___Obtaining blood below the IV line of a patient.


___Mixing of anticoagulated blood by shaking the tube
gently
5-8 times.
___Obtaining a blood sample from a patient who
underwent
blood transfusion just 30 minutes
ago.
___Drawing blood from the feet.
___Applying tourniquet for two minutes.
___Avoiding to touch again a thoroughly cleaned
puncture
site.
___Collecting SST before a yellow vacutainer in onemovement technic.
___Collecting PST before a dark green heparinized
tube in one-movement technic.

Quick
Quiz

Which of the following is an unnecessary step


in the proper phlebotomy procedure?
a. recapping the needle after use
b. placing bandage over the punctured
site
c. washing the hands prior to
venipuncture
d. sterilizing the puncture site

Which vein cannot be used in obtaining


venous blood in an adult?
a. radial vein b. jugular vein
c. ankle vein d. dorsal hand vein

Arterial puncture

Arterial blood is oxygenated blood. It is


uniform in composition throughout the body.

Arterial blood is used to measure oxygen


tension, carbon dioxide tension and
blood pH.

Blood gas analyses (BGA) are critical to patients


with pulmonary problems, oxygen
therapy, cardiovascular problems and
those undergoing major operations.

Sites of arterial puncture

Radial arteries
Brachial arteries
Femoral arteries

Radial and brachial arteries are the preferred


sites.

Each site of arterial puncture uses different gauge of


needles:

In collecting arterial blood, needle is positioned at


different angles in each site:

Brachial artery- 18-20 gauge


Radial artery- 23-25 gauge

Brachial artery- 45-60 degrees


Femoral artery- 90 degrees

Heparin is used as anticoagulant for arterial puncture.

Complications of arterial puncture

Hematoma due to increased pressure in the


artery

Restriction of blood flow due to reflex


constriction (arterial spasm)

Temporary discomfort (aching, throbbing,


tenderness, sharp sensation, and
cramping)

Thrombosis, hemorrhage and infection

Considerations

Intense care should be administered to patients undergoing


arterial puncture.

Sites that are irritated, edematous, near a wound or in an


area of arteriovenous (AV) shunt or fistula should not be
selected for arterial puncture.

Samples are placed in ice water or other coolant


(temperature of 1-5C) to minimize leukocyte

consumption of oxygen.

Capillary blood may be a suitable substitute for

arterial blood determination of pH and


pCO2 provided that the site must be warmed prior to

collection. Warning increases blood flow through capillaries


and arterioles and result in arterial-rich blood.

Quick Quiz

Which test does not require an arterial blood


sample?
a. oxygen tension b. blood pH
c. pCO2 determination d. plasma hemoglobin

Which statement is false regarding arterial blood?


a. Arterial blood collection always requires the
use of anticoagulant.
b. Arterial blood is a must in blood gas analyses.
c. Arterial blood collection requires a glass
syringe to minimize leukocyte consumption of
gases.
d. Arterial blood has lower pCO2, therefore, is
less acidic than venous blood.

Skin puncture/Capillary puncture


Capillary blood is an admixture of venous, arterial
and capillary blood and may contain tissue
fluids.
Skin punctures are suitable in pediatric patients,

obese patients with thrombotic tendencies


and severe burns. It is often preferred to

geriatric patients due to thinness and less


elasticity of skin.

Sites of skin puncture

Infants
Lateral plantar heel surface
Median plantar heel surface
Children
Plantar surface of the big toe
Plantar surface of the last digit of the 2 nd, 3rd or 4th fingers
may be used in older infants
Lateral side of the finger adjacent to the nail

Capillary blood can be collected for analysis of blood


gas (site must be pre-warmed before collection).

The best method of arterial blood collection in newborn


is the umbilical artery catheter.

Skin puncture site must be warmed before collection to


increase the blood flow (arterialization).

Thermal using dry heat or paper towel with


warm water (39-42OC)
Mechanical by flicking with index finger until
flushing is observed
Chemical using Trafuril paste or Histamine creams

Arterial blood collected from capillary


puncture may yield unreliable results if:
Systolic pressures is less than 95
mmhg
Cardiac output is severely restricted
Vasoconstriction is present

Puncture depth ranges from 0.85 to 2.00


mm deep and 1.75-3.00 mm in length.

Quick Quiz

Which test requires finger skin puncture?


a. Rumpel-Leede b. Schryver and
Waugh
c. Ivy Bleeding Time d. Lee-White

Which blood collection technic requires an


arm maneuver known as Allen test to check
for collateral circulation?
a. phlebotomy b. arterial puncture
c. skin puncture d. peripheral
puncture

VACUTAINER/EVACUATED TUBES

These are tubes for blood collection which


are color-coded based on the anticoagulant
present. They come in various sizes; 2, 5, 7,
and 10 ml.

Blood is drawn in this order: Blood culture


tubes, red top, blue top, green top, lavender
top and gray top

COLOR

ADDITIVE

Lavender

Ethylenediaminetetraacetic
chelates calcium
Acid (EDTA)
* Versene (disodium salt)
* Sequestrene (dipotassium salt)

Red

None

Red Gray or

None but contains

Red Black

separator material
and serves as a
barrier between cells
and serum

Yellow

Citrate dextrose

Green

Heparin (Na+, Li+,


or, NH4+)

ACTION

Allows blood to clot

Allows blood to clot

preserves RBCs

inhibits thrombin
activation

USE
hematologic assays
lead assay, CEA
Determination and
cell counts
Most chemistry,
immunologic and
blood bank tests
Most chemistry tests

Blood culture

ammonia CO-Hb
and methemoglobin

COLOR ADDITIVE

Orange Thrombin
Blue

ACTION

USE

accelarated clot

STAT serum tests

Buffered citrate binds calcium


like PT & APTT

Black Buffered sodium

Coagulation assays

binds calcium

Westergren ESR

citrate

Gray

NaF/K2C2O4
Inhibits glycolytic
enzyme enolase and
determination
act as anticoagulant
Iodoacetate inhibits glycolytic
3-phosphate
dehydrogenase

Glucose

Glucose

enzyme glyceraldehyde

determination

Anticoagulant Interference

Dilution errors especially oxalates which are highly osmotic

Inhibition of plasma enzyme activities activities especially


with fluoride which is an enzyme poison, EDTA which
chelates metallic enzymes activators. Oxalate inhibits AMS,
LD and ACP, and citrate which inhibits AMS

Oxalates, citrate and EDTA lower plasma calcium levels

False increase in electrolyte analyses due to the


anticoagulants in the salt form

If multiple tubes are needed, the proper order of draw to avoid


cross contamination and erroneous results is as follows:
1st -Blood culture vials or bottles, sterile (yellow or yellowblack top)
2nd - Coagulation tube (light blue top) NOTE: If just a routine
coagulation assay is the only test ordered, then a single light
blue top tube may be drawn. If there is a concern regarding
contamination by tissue fluids or thromboplastins, then one may
draw a non-additive tube first, and then the light blue top tube.
3rd - Non-additive tube or Serum tube
Last draw - additive tubes in this order:
1. SST (red-gray or gold top). Contains a gel separator and clot
activator.
2. Sodium heparin (dark green top)
3. PST (light green top). Contains lithium heparin
anticoagulant and a gel separator.
4. EDTA (lavender top)
5. ACDA or ACDB (pale yellow top). Contains acid citrate
dextrose.
6. Oxalate/fluoride (light gray top)

Quick Quiz

The light-sensitive substance that


provides the normal straw color of serum
and plasma is
a. urochrome
b. bilirubin
c. flavin compounds
d. betacarotene

The vacutainer required in the


measurement of serum ammonia is
a. green top
b. red top
c. blue top
d. lavender top

Specimen Handling and Processing


Serum

20-30 minutes is the ideal clotting time


Generally more preferred than plasma
1.
2.
3.

Interfering substances are coprecipitated during clotting such as LPL


Optically clearer
Free from anticoagulant interference

Must ideally reach the laboratory within 45 minutes


Agitation must be avoided during transport
Use amber containers for photolabile substances
Transport in ice (4 deg C) those specimens for BGA,

renin, enzymes and catecholamines

Specimen Interference

Lysis of cells or Laking (Hemolyzed serum)

Results in leakage of intracellular substances

Lysis of RBCs is called laking or hemolysis which may


occur in vivo or in vitro

In vitro hemolysis is more common which may be due to:


1. Use of vacuum tubes
2. Vigorous mixing
3. Use of too narrow or too wide needle bores
4. Effect of alcohol
5. Centrifugation and separation steps
Hemolysis is visible only not until a 200 mg/L of

hemoglobin level in present

Icteresia (Icteric serum)

Intensely yellow serum sample due to elevated


bilirubin value

Jaundice in a patient is caused by a bilirubin


level of greater than 430 M (25 mg/L)

Bilirubin interferes with tests using dyes and


turbidity tests

Interference due to bilirubin may be minimized


by sample blanking or dual wavelength
method known as the Allen correction method

Lactescence (Lipemic serum)

Obtained normally after a meal due to elevated chylomicrons

Characterized by milky or highly turbid serum

Lactescence appears when the TAG level reaches 4.6 mM (4g/L)

Errors due to lipemia may be corrected by ultracentrifugation of


the serum sample

Grounds for rejecting a specimen


Inadequate sample identification
Insufficient

collection

volume of specimen

Inappropriate

collection tube

Hemolysis
Improper

transportation

Interferences

Last Quick Quiz

Which sample below will likely require


ultracentrifugation to minimize sample
interference?
a. icteresia
b. lipemia
c. laking d. all of the above

Prompt separation of the clot from serum


should always be observed because this
substance substantially decrease by 5% per
hour if serum is left in contact with clot. This
substance is
a. sodium b. albumin
c. glucose d. phosphate

QUALITY MANAGEMENT
Quality Assurance (QA)

includes maneuvers encountered in the


analytic, pre-analytic and postanalytic phases of laboratory testing

Pre-analytical phase includes:


test ordering
specimen collection
transport of the specimen in the laboratory

Pre-analytical phase includes:


processing

of specimen
entering patient information
centrifuging
separating aliquoting specimen
delivery to proper laboratory location

Analytical phase includes


specimen analysis (manual or automated)
use of commercial controls
record keeping
Post-analytical phase includes
reporting out results of analysis (manual or
computerized)
physician contact

Post-analytical phase also includes


monitors

quality performance starting from


the ordering of a laboratory determination
to its reporting, interpretation of results,
and then application to patient care

involves

total quality control which requires


constant attention of all involved in the
laboratory testing

Quality Control (QC)

is concerned with the analytic phase of QA

monitors the over-all reliability of


laboratory results in terms of accuracy

and precision

relies on the diligent and persistent execution of the


following QC associated activities:
assay

of control samples
instrument maintenance
statistical data analyses
proficiency testing survey

has two major types


External QC (Interlaboratory QC) was
established by Belk which monitors primarily the
accuracy of laboratory tests; the use of Youden
plots

Internal QC (Intralaboratory QC) was

established by Levey which primarily monitors the


day-to-day performance of laboratory tests precision assessment. Intralaboratory QC can
be based either from the results of control
specimens or on the results of patient specimens;
the use of Levey-Jennings chart

Quick
Quiz
Which
QC chart is a graphic representation
of the acceptable limits of variation in the
results of an analytical method?
a. Gaussian
b. Youden
c. Levey-Jennings d. Cusum

Which of the following has as its purpose to


promote the incorporation of principles of
quality management and QA into daily work
routines?
a. ISO 9000
b. NCCLS
c. CAP
d. NIST

Errors
Analytical errors are usually systematic
errors or determinate errors that are
caused by some factors in the analytical
system such as erroneously calibrated
pipettor, deteriorating reagent and
fluctuating electrical current.
Personnel or operator errors are usually
called random errors or indeterminate
errors that usually affect several analyses.
Examples are mislabeling the specimen,
wrong number entry and instability of
needle due to electronic component of
instrument.

Interpretation of the LeveyJennings Chart


Substance
+3SD
+2SD
+1SD
Mean
-1SD
-2SD
-3SD

Shift
Dispersio
n
Trend

Days of a particular month

Trend suggests a systematic drift or error.


Values move continuously away from the mean
in just one direction. Among its causes include
deteriorating reagents, changes in standard
concentration or failing instrument.
Shift also suggests systematic error. Seen as
an abrupt change from the established mean
and continue in a linear fashion parallel to the
mean.
Increased dispersion suggests random error.
High or low outliers are frequently observed.
Causes of this are variations in operation of
instrument, interfering substances, electronic
fluctuations, and clerical errors.

Quick Quiz

Which of the following terms refers to


deviation from the true value caused by
indeterminate errors inherent in every
laboratory management?
a. random error b. standard error of the mean
c. parametric analysis d. nonparametric
analysis
When comparing a potential new test with a
comparative method in order to bring a new
method into the laboratory, one observes error
that is consistently affecting results in one
direction. This type of error is
a. systematic error
b. random error
c. constant systematic error d. proportional
random error

Westgard Multi-Rule
Technic
Rule
Meaning
1:2s = one control observation exceeds the control

limit set at
M2SD; warning rule
1:3s = reject when one observation exceeds M3SD;
suggests
random error
2:2s = reject when two consecutive observations
exceed the same
M+2SD or the same M-2SD;
suggests systematic
error
R:4s = reject when one control observation in the run
exceeds its
M+2SD and another exceeds M-2SD;
suggests
random errors
4:1s = reject when four consecutive control
observations exceed
the same M+1SD limit or
the same M-1SD limit; suggests
systematic error
10:Mean= reject when 10 consecutive control
observations fall on one side of the mean; suggests
systematic error

Quick
Quiz
What
does the preparation of a Levey-Jennings

chart for any single constituent of serum require?


a. analysis of control serum over a period of 20
consecutive days
b. 20-30 analyses of the control serum, on 1 day,
in one batch
c. analyses consistently performed by one person
d. weekly analyses of the control serum for 1
month

In addition to utilizing Levey-Jennings charts, what


other criteria should be applied to interpret
internal QC data?
a. Westgard multirule
b. Cusum
c. linear regression d. Youden

Accuracy and Precision


Accuracy is the extent to which the mean

measurement is close to the true value.


The accuracy of the method is reflected by its ability
to obtain the same values of the reference
samples of known concentration. Expressed
as % absolute error.
% absolute error =
true value

true value actual value x 100%

Precision is the reproducibility of a laboratory

determination when it is run repeatedly under identical


conditions
precision is commonly expressed in terms of
standard deviation (SD), variance or
coefficient of variation (CV)

Standard deviation =

(M x)2

n-1
Variance = (SD)2
Coefficient of variation =
100%
M

SD x

Reliability refers to the ability of a test to


maintain its accuracy and precision
for an extended period of time.

Quick Quiz

Which of the following terms refers to


the closeness with which the measured
value agrees with the true value?
a. random error
b. precision
c. accuracy
d. variance

Given that a method mean is 25 mg/dl


and the SD is 1.2 mg/dl, what would be
the CV?
a. 2.1%
b. 2.4%
c. 4.8%
d. 9.6%

SD is a measure of dispersion of the values around the mean


and in normal or Gaussian distribution,

68% of the values fall within +/-1 SD around the mean


95% of the values fall within +/-2 SD around the mean

99.7% of the values fall within +/-3 SD around the mean

most laboratories choose the 95% confidence limit in


expressing precision

the median is the value that is middlemost in an array of


numbers while the mode is the value that occurs most
frequently.

In a Gaussian distribution, the mean, median and mode


are very close in value as shown by its bell-shaped curve.

Quick Quiz

In a QC program the confidence interval


has been set at 95%. How many test
results are expected to fall beyond the
established limits?
a. 1 in 5 b. 1 in 10
c. 1 in 20 d. 1 in 95

What percentage of values will fall


between 2SD in a Gaussian distribution?
a. 34.13% b. 68.26%
c. 95.45% d. 99.74%

Sensitivity is the ability of the test to detect the

smallest amount of the analyte in a


solution or sample. It expresses the ability of the
test to be positive in the presence of the analyte or the
disease. A highly sensitive test is characterized by a
decreased probability of obtaining false
negative results.

Specificity refers to the ability of the test to detect


analyte without detecting other analytes
that are also present in the sample. It
expresses the ability of the test to be negative in the
absence of the analyte or the disease. A highly
specific test leads to a decreased probability of
obtaining false positive results.

Control is a solution (usually pooled serum


samples) whose constituents are diverse
but are known (a range of values per
analyte). This can be run simultaneously
with the Test to check, verify or validate
the accuracy of the results.
Standard is a solution of a particular
analyte of known characteristics and
known value (exact concentration). It is
used as reference for the calculation of
the value of the Unknown.

Quick Quiz

To determine the predictive value of a positive


test, all of the following parameters must be
known except
a. sensitivity b. specificity
c. disease prevalence d. precision

Which activity will not check on the accuracy of


the results obtained?
a. make a Youden plot b. use of abnormal
control
c. use of normal control d. use of a calibrator

(standard)

Figures of Merit of
Analytical Methods

Major

1. Accuracy
2. Precision
3. Limit of detection
4. Applicable concentration
range or
linear range
5. Sensitivity
6. Selectivity

Minor

1. Speed
2. Ease and convenience
3. Skill required of the
operator
4. Cost of analysis and
availability of equipment
5. Per sample cost

End