INSTRUMENTATI

ON
LESSON
Group 6
 01
CENTRIFUG
ES
 Centrifuges
● Checks centrifuge speed for accuracy: TACHOMETER/
STOBE LIGHT
● Checks timer of centrifuge: STOPWATCH
● Disinfection: EVERY WEEK
● Calibration: EVERY 3 MONTHS
● Hazard associated with the breakage in centrifuge: AEROSOL
HAZARD
Types of Centrifuges
HORIZONTAL-HEAD
CENTRIFUGE/
SWINGING BUCKET
• Tubes when spinning:
HORIZONTAL
• Tubes when not moving:
VERTICAL
ANGLE-HEAD CENTRIFUGE/
FIXED ANGLE
• Tubes are at fixed angle (25 to 40
degree) when rotating
• Ex. Microhematocrit centrifuge
CYTOCENTRIFUGE
• Uses a very high torque and low-
inertia motor to spread a
monolayers of cells rapidly across
a special slide for critical
morphologic studies
• Slowest centrifuge

ULTRACENTRIFUGE
• High speed centrifuge
• Fastest centrifuge
 02

PIPETTE
S
 Pipettes
TO DELIVER TO CONTAIN
Deliver or dispenses stated volume Contains particular volume but does not
dispense exact volume

BLOW-OUT SELF DRAINING

With etched ring No etched ring
Last drop should be expelled Contents drain by gravity

TRASNFER PIPET MEASURING PIPET AUTOMATIC PIPET

Volumetric Serologic Air Displacement
Ostwald-Folin Mohr Positive Displacement
Pasteur Micropipet Dispenser & Dilutor
dispense
 TRANSFER PIPET MEASURING PIPET
- Dispense one volume without - Capable of dispensing several
further subdivisions different volumes

BLOW-OUT OSWALD-FOLIN SEROLOGICAL PIPET

- With etched ring - For viscous fluid (whole blood) - With graduation marks down
- With bulb closer to the the tip
delivery tip - For serial dilutions and
measuring reagents
SELF-DRAINING VOLUMETRIC PIPET MOHR PIPET
- No etched ring - For non-viscous or aqueous - No graduation mark to the tip
solutions - Tip should not be allowed to
- With bulb closer to the center touch the vessel while pipet is
draining

PASTEUR PIPETTE Micropipette

- No calibration marks For volumes ranging from 1 to
- Used to transfer solutions 1,000 uL
without consideration for
specific volumes
 Pipettes
AUTOMATIC PIPET
- Most routinely used in clinical chemistry laboratory
AIR DISPLACEMENT POSITIVE DISPENSER & DILUTOR
PIPET DISPLACEMENT PIPET DISPENSER
- Relies on piston for - TIPS ARE REUSABLE - Obtain the liquid from a
creating suction to common reservoir and
draw the sample into dispense it repeatedly
a disposable tip (TIPS
CAN ONLY BE USED
ONCE)
- Piston does not come
in contact with the
liquid
Pipettes
 03
CALIBRATION
& AUTOMATION
 Calibration
● GRAVIMETRIC METHOD
- Most accurate method
- Delivering and weighing a solution of known specific
gravity (WATER)
● SPECTROPHOTOMETRY
- Secondary method
- Absorbance of potassium dichromate or p-nitrophenol
delivered
 TYPES OF AUTOMATION
ANALYSIS
SEQUENTIAL Analyzes multiple tests one after
another on a given specimen
PARALLEL Analyzes more than one test
concurrently on a given clinical
specimen
RANDOM Performs any test on any sample in
any sequence
BATCH All samples are loaded at the same
tine, and a single test is conducted on
each sample
Other Terms
Stand Alone Instrument from a single discipline with
automated capability
Automated Stand Instrument from a single discipline with additional
Alone intern automated capability (e.g., auto-repeat,
auto-dilute
Modular Workcell At least two instruments from a single discipline
with one controller
Multiple Platform Instruments able to perform tests from at least
two discipline
Integrated Modular At least two analytical modules supported by one
System sample and reagent processing and delivery
system
Pneumatic Tube Transports specimens quickly from one location
System to another
Other Terms
Throughput Maximum number of tests generated per hour

Turnaround Amount of time to generate one result

Bar Coding Mechanism for patient/sample identification; used for

reagent identification by an instruments
Dead Volume Amount of serum that cannot be aspirated

Carry-over Contamination of a sample by a previously aspirated

sample
Reflex Testing Use of preliminary test results to determine if additional
tests should be ordered or cancelled on a particular
specimen; performed manually or automated
Dwell Time Time elapsed from the initiation of the test up to the
completion of analysis
 BASIC APPROACHES TO
•
AUTOMATION
All three can use batch analysis
• Only DISCRETE analyzers offer random access or stat capabilities
CONTINOUS FLOW CENTRIFUGAL DISCRETE
- Liquids are pumped - Use the force generated by - Houses samples and
through a system centrifugation to transfer reagents in separate
continuous tubing and then contain liquids in containers and each
- Separating/cleaning separate cuvettes for sample reaction is
media: AIR BUBBLES measurement at the compartmentalized
- Mixing accomplished perimeter of a spinning - Most popular and versatile
through the used of rotor analyzer
COILING TUBE - Most capable of running - With random-access
- Major drawback: Carry- multiple samples one test capability
over problems and at a time
wasteful use of - Major advantage: Batch
continuously flowing analysis
reagents
REFLECTANCE PHOTOMETRY
● It is the measurement of light reflected from solid surfaces.
● A reflectometer is used to measure analytes
● Kodak Ektachem (Vitros) and automated urine dipstick
readers
Spreading layer
LAYERS OF DRY SLIDE TECHNOLOGY
The point of contact with sample

Scavenger layer Between spreading layer and reagent

layer; removes materials present in the
sample which might interfere with the
reaction
Reagent layer Contains all the chemicals necessary
for the reaction
Indicator layer Optional layer; contains a dye

Support layer Clear plastic on which the entire slide is

constructed
POINT OF CARE
TESTING (POCT)
- Performing diagnostic tests
outside the main laboratory and
at/or near patient care areas
- Also known as decentralized,
bedside, ancillary, extra-
laboratory, near patient,
physician’s office and alternative
site testing
- Performed by clinical staff
(nurses, doctors, RTs, etc.)
rather than laboratorians. Also it
can be performed by patients at
home
- Major advantage: Faster
delivery of results
- May be used in ICU or
emergency department
COMMON POCT TEST: urine
reagent strip, glucose,
electrolytes, blood gases,
ACT, PT, APTT, hemoglobin
- QC testing for bench top
and/or multitest analyzer: at
least once per shift = THREE
TIMES A DAY
 04

BEER’s
LAW
 BEER’s Formula

• LAW
States that absorbance (optical
density) is DIRECTLY
Formula:
A = abc
PROPORTIONAL to A = log10 100 / %T,
concentrations A = 2 - log10 %T
• States that the concentration of
the analyte is INVERSELY “A” (absorbance)
PROPORTIONAL to the “a” (absorptivity)
logarithm of light transmitted “b” (light pattern of the solution
in cm)
ABSORBANCE – Amount of light “c” (concentration of the
blocked by the solution substance)
TRANSMITTANCE – ratio of %T (transmittance)
transmitted light to the incident light
 BEER’s LAW
AREA WHERE BEER’S LAW CANNOT
BE FOLLOWED
Unless the absorptivity is constant over
the range of wavelength being used
If two or more chemical species are
absorbing the wavelength of light being
used, each with different absorptivity
If the absorption of a fluorescent solution
is being measured
ARE WHRE BEER’S LAW CAN BE
FOLLOWED
If the incident radiation on the substance
of interest is monochromatic
If the solvent absorption is insignificant
compared with the solute absorbance
If the solute concentration is within linear
limits
If a chemical reaction does not occur
between the molecule of interest and
another solute or solvent molecule
SPECTROPHOTOMETER
 Incident light Transmitted light
0.43
specific wavelength (color) the solute absorbs some digital display of absorbance
that the solute will absorb 1 M CuSO4 of the incident light
 SPECTROPHOTOM
●
ETRY
Principle: chemical reaction produces colored substance that absorbs
light of specific wavelength
● Amount of light absorbed is DIRECTLY PROPORTIONAL to the
concentration of analyte

COMPONENTS OF SPECTROPHOTOMETRY
1. Light source – provides the radiant energy
UV (4 nm to 400 nm) Deuterium or mercury Quartz (silica) cuvet
Visible light (400 nm to Tungsten Borosilicate
700 nm)
Infrared (750 nm to 0.3 Tungsten Quartz (silica) cuvet
cm)
NANOMETER: unit of measurement for wavelength of radiant energy (light)
Wavelength is INVERSELY PROPORTIONAL to the amount of energy thus the
longer the wavelength, the lower the energy and vice versa
 SPECTROPHOTOME
2. TRYthe stray light emitted by the
Entrance slit – minimizes
lamp and prevents scattered light from entering the
monochromator
3. Monochromator – isolates specific wavelengths of light
4. Exit slit – controls the amount of light that passes through
the cuvette
5. Cuvette/analytical cell/absorption cell – holds the
solution whose concentration is to be assayed
6. Photodetector – measures the intensity of the light from
the solution
7. Readout device – numerically presents the absorbance or
percent transmittance
SPECTROPHOTOM
ETRY
 BLANK CORRECTION/
BLANKING
• Ensures the accuracy of the results by eliminating interfering
substances inherent with the sample or the reagent

Calibration blank is a blank used to ensure that the measurement

device gives a zero signal when there is no sample present. This can
be an electronic adjustment of the signal intensity in the absence of
sample.
Reagent blank is a solution that contains all the same components
(matrix) as the sample solution, but no known analyte materials. It
corrects absorbance caused by the color of reagents.
Sample blank – used to correct mom-specific absorbance of
substances in the sample
Water blank – sets the spectrophotometer at zero absorbance
 OTHER
METHODS
 OTHER METHODS
1. Flame Emission
Photometry (FEP)
• It measures the light emitted by a
single atom burned in a flame.
• Principle: Excitation of electrons from
lower to higher energy state.
• Light source: Flame (also serves as
the cuvette)
• It is used for the measurement of
excited ions [Sodium (yellow),
Potassium (purple), Lithium (red)]
 OTHER METHODS
2. Atomic Absorption Spectrophotometry (AAS)
• It measures light absorbed by the ground-
state atoms.

• Light source: Hollow cathode lamp,

atomizer, flame, mixing chamber, chopper,
monochromator, detector, readout device
• It is used for the measurement of
unexcited trace metals (Ca&Mg). More
sensitive (100x) than FEP; it is accurate,
precise and very specific
 OTHER METHODS
3. Fluorometry
• It determines the amount of light emitted by a
molecule after excitation by electromagnetic
radiation.
• Absorb light of specific wavelength and emit
light of longer wavelength
• Uses: Measurement of porphyrins,
magnesium, calcium, drugs, hormones
• Primary monochromator: Excitation
• Secondary monochromator: Emitted
• Advantage: SENSITIVE AND SPECIFIC
(1000x more sensitive than most
spectrophotometric methods)
• Disadvantage: very sensitive to
environmental changes
4. Turbidimetry
• Measures reduction in light transmission
by particles in suspension
• Principles: Determines the amount of
light blocked by a particulate matter in a
turbid solution.
• Used for Proteins in urine and CSF
5. Nephelometry
• For measuring the amount of antigen-
antibody complexes (proteins)
• Principle: Determines the amount of
scattered light by a particulate matter
suspended in a turbid solution
• Light is measured at angle from light source
(light scattering)

6. Chemiluminescence
• Chemical reaction that produces light
• Usually involves oxidation of luminol,
acridinium esters, or dioxetanes
• Used for IMMUNOASSAYS
7. Potentiometry
• Measures electrical potential due to the
activity of free ions; change in voltage
indicates activity of each analyte
• Used for pH and pCO2 test
8. Amphelometry
• It measures current flow produced by
oxidation reaction
• Uses: pO2, glucose, chloride, and
peroxidase determinations
REFERENCES
• Clinical Chemistry: Principles, Techniques and Correlations 8th
ed by Michael L. Bishops
• Clinical Chemistry: A Fundamental Textbook 2nd ed by Donald
Calbreath
• Clinical Chemistry Review Handbook by Maria Teressa
Rodriguez
• Linne & Ringsrud’s Clinical Laboratory Science 7th ed by Mary
Louise Turgeon
THANK
YOU!