CLINICAL CHEMISTRY

LECTURE / FIRST SEMESTER

UNIT 3: ANALYTIC TECHNIQUES
Topic Outline:
 Spectrophotometry
 Beer’s Law
 Components of a Spectrophotometer
 Quality Assurance
 Atomic Absorption Spectrophotometry
 Fluorometry
 Chemiluminescence
 Turbidimetry and Nephelometry
 Laser Applications
FIGURE 5-1. Electromagnetic radiation—relationship of energy
and wavelength.
Spectrophotometry
Electromagnetic Radiation Wavelength:
- described as photons of energy traveling in waves  Visible Region: 400 - 700 nm
 Ultraviolet: < 400 nm
- can take several forms, the most recognizable being
 Infrared Region: > 700 nm
light and radiant energy
- Other Types: Gamma rays and X-rays, microwaves, The higher the wavelength, the lower is the energy. The
radio frequency radiation, and ultraviolet radiation lower the wavelength, the higher is the energy.

Wavelength (λ)
- the linear distance between any two equivalent points
on a successive wave
- analytes in the laboratory are measured based on
their maximum absorbance or based on their own wavelengths
- Example: Bilirubin is an analyte that is maximally
absorbed at 450nm, which means the wavelength must be set at
450nm in order for the analyte to be measured
- unit used in the visible spectrum is nm Beer’s Law
- also known as Beer-Lambert’s Law
- states that the concentration of a substance is directly
proportional to the amount of light absorbed or inversely
proportional to the logarithm of the transmitted light
- Absorbance and % Transmittance

The relationship between wavelength (λ) and energy E is

described by Planck’s formula.

E = hv

where h is the Planck’s constant (6.62 x 10 -27 erg sec)

and v is frequency.

Frequency
- the number of oscillations of the waveform in a second
*Oscillation - any changes that may occur in a second

Frequency is inversely proportional to the wavelength.

T = radiant energy transmitted / transmitted light
Energy of electromagnetic radiation is inversely I = radiant energy incident on the sample / incident light
proportional to the wavelength.

ε = molar absorptivity
b = the length of light path through the solution
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LECTURE / FIRST SEMESTER
c = the concentration of absorbing molecules
The molar absorptivity and the length of light path is Characteristics:
constant therefore, the absorbance is directly  Nominal Wavelength - represents the wavelength in
proportional to the concentration of absorbing nm at peak transmittance
molecules.  Spectral Bandwidth (or FWHM) - locate the 1/2 peak
height of peak transmittance and it is the range of the
wavelength
Spectrophotometer
* Full Width at Half Peak Maximum
- used to measure the light
transmitted by a solution to determine  Bandpass - total range of wavelength transmitted
the concentration of light-absorbing
substance in the solution.

Components of a Spectrophotometer
1. Light Source
2. Monochromator
3. Sample Cell or Cuvet
4. Photodetector
5. Meter or Read-Out Device
Three Types:
A. Filters
Light Source
- isolate monochromatic light
- provides polychromatic light: light with several
- simple, inexpensive, and useful
wavelengths and capable of striking a beam of light into the
- Example: Interference and absorption filters
monochromator and the beam of light produces several
wavenlengths
B. Prism
- provides energy that the sample will modify or
- can be rotated, allowing only the desired wavelength
attenuate by absorption
to pass through an exit slit
- dependent on wavelength requirement
- Incandescent Tungsten or Tungsten-Iodide Lamp -
C. Diffraction Gratings
visible and near-infrared regions
- most commonly used; contain parallel grooves which
- Deuterium Lamp and Mercury Arc Lamp - UV
aids in selecting the desired wavelength to pass through
region
the exit slit

Two Types: Sample Cell or Cuvet

A. Continuum - may be round or square and must be made of material
- wide applications in the laboratory that is transparent to radiation
- emits limited number of discrete lines or bands of - used to hold samples; path length is 1 cm
radiation - Double-beam Spectrophotometers - two cuvets (for
- Examples: the sample and for the solvent)
 Tungsten - Visible Region
 Deuterium - UV Region Three Types:
 Xenon - Visible and UV Regions A. Fused Silica or Quarts - UV region
B. Alumina-silicate Glass - 350 - 2000nm
B. Line C. Plastic Cuvet - visible region
- Emits a few discrete lines or bands of radiation
- Examples: Photodetector
 Mercury and Sodium Vapor Lamps - UV and - converts the transmitted radiant energy into an
Visible Regions equivalent amount of electrical energy
 Hollow Cathode Lamp - atomic absorption
spectroscopy / spectrophotometry Four Types:
 Electrodeless Discharge Lamp - another light A. Barrier-layer Cell or Photocell
source for AAS - least expensive; temperature sensitive
- machine needs to be warmed-up before testing
Monochromator - composed of selenium on a plate of iron
- isolates individual wavelengths of light coming from - used mainly in filter photometers which uses filters as
the monochromatic light provided by the light source monochromators
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B. Phototube Double-Beam Spectrophotometer

- contains cathode and anode enclosed in a glass tube
- has photosensitive material that gives off electrons
when light energy strikes it

C. Photomultiplier Tube (PMT)

- most common type
- 200 times more sensitive than the phototube
- highly sensitive to UV and visible radiation The light source provides energy or light and it strikes
the monochromator through the entrance slit. The
D. Photodiode monochromator is capable of selecting individual wavelengths of
- not as sensitive as Photomultiplier tube but with light provided by the light source. From the monochromator, a
excellent linearity and speed beam of light passes through the sample cuvet through the exit
slit and through the reference cuvet. Some of the light will be
*Linearity - signal transmitted by the photodetector into absorbed by the molecules present in the sample and the
the read-out device is a real reflection of the concentration remainder strikes the photodetector, a photomultiplier tube
of the analyte of interest capable of transmitting the radiant energy passed by the sample
cuvet into the equivalent electrical signal. The signal will first be
Meter or Read-Out Device converted into a voltage and into the digital signal using the A/D
- displays output of the detection system converter. After that, the digital signal is processed to produce an
- Examples: absorbance reading which will be displayed by the read-out
 Digital Meters, d’Arsonval meters, recorders, device or meter.
Light-Emitting Diodes (LEDs), Cathode-Ray
Tubes (CRTs), and Liquid Crystal Displays Two Instrument Designs:
(LCDs) 1. Double Beam in Space
- uses 2 photodetectors
Single-Beam Spectrophotometer
- one measurement at a time at one specified 2. Double-Beam in Time
wavelength - uses 1 photodetector; chopper is used to pass the
- the wavelength of the analyte to be measured must be monochromatic radiation through the sample cuvet and
known in advance then to the reference cuvet
 Chopper - a device that rotates or breaks up
radiation beams

Quality Assurance in Spectrophotometry

1. Wavelength or Photometric Accuracy
- implies that a photometer is measuring at the
wavelength that it is set to
- Special glass-type optical filters - didymium glass
(600nm); holmium oxide (360nm)
The light source provides polychromatic light. The beam
of light coming from the light source exits through the entrance 2. Absorbance Check
slit, and enters the monochromator through the entrance slit. In - using glass filters or solutions that have known
the monochromator which comes in the form of grating will have absorbance values for a specific wavelength
the capacity to isolate the individual wavelengths of light coming
from the polychromatic beam of light. A beam of light exits 3. Linearity
through the exit slit and strikes the cuvet containing the sample. - the ability of a photometric system to yield a linear
From the cuvet, some of the light is absorbed by the molecules relationship between the radiant power incident upon its detector
present in the sample and the remainder strikes now the and the concentration
photodetector which is the photomultiplier tube. The role now of - optical filters or solutions
the photomultiplier tube is to convert the radiant energy
transmitted from the sample cuvet into the equivalent electrical 4. Stray Light
signal. After which, the electrical signal is converted to a voltage - any light that impinges upon the detector that does not
and a digital signal using the Analog to Digital Converter. The originate from a polychromatic light source
digital signal is processed to produce now the reading displayed - interference; Causes: scratches on optical surfaces
by the read-out device or meter.
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- Special Cutoff Filters - eliminate all radiation of the sample into the monochromator. The monochromator will
wavelengths beyond the one of interest have to isolate the desired wavelength of interest for that specific
Atomic Absorption Spectrophotometry (AAS) analyte to be measured and a beam of light will have to strike the
 Atomic Absorption Spectrophotometer photodetector which comes in the form of photomultiplier tube.
- measures concentration by detecting the absorption of The PM tube will convert the radiant energy transmitted by the
electromagnetic radiation by atoms rather than by molecule monochromator into an equivalent electrical energy into the
- Atomization using heat to break the chemical bonds voltage and into a digital signal displayed by a read-out device or
to produce a free unexcited atom meter.
- Atoms present in the sample is the reflection of the
concentration of the analyte of interest Flameless Atomic Absorption Spectrophotometry
- Light Source = hollow-cathode lamp; electrodeless - uses an electric furnace to break chemical bonds
discharge lamp (electrothermal atomization)
- Chopper = modulate the light beam, controls the - uses a tiny graphite cylinder to hold a sample and an
intensity of light coming from the hollow-cathode lamp or the electric current passes through the cylinder walls and heats now
electrodeless discharge lamp into the flame which serves as the the entire units to atomize the sample
sample cell or cuvet
- Photodetector = photomultiplier tube Fluorometry
1. Filter Fluorometers
 Sensitive and Precise - measure the concentrations of solutions that contain
 Application: To measure concentrations of trace metals fluorescing molecules
- used for analytes that have the capacity to fluorease
Single-Beam Atomic Absorption Spectrophotometer
- ensure that atoms are produced because the amount 2. Light Source
of light absorbed by atoms present in the sample is directly  Mercury - Filter Fluorometers; monochromators come
proportional to the concentration of analyte of interest in the form of filter
- The flame is the sample cell or the sample cuvet in the  Xenon Arc - Spectrofluorometers; monochromators
instrument come in the form of grating or filters
- Most Common Burner: Premix Long-Path Burner
3. Photodetector
Basic Components: - Photomultiplier tube
1. Light Source - comes in the form of a hollow-cathode lamp
or electrodeless discharge lamp Basic Filter Fluorometer
2. Copper - controls the intensity of light from the light source Components:
into the atoms present in the sample 1. Light Source - usually mercury
3. Monochromator 2. Attenuator / Mechanical Attenuator - controls the beam
4. Photodetector of light from the light sourcer to the primary filter
5. Read-out Device or Meter 3. Two Monochromators - Primary Filter and Secondary
Filter
4. Sample Holder / Cuvet
5. Photodetector
6. Read-out Device

The sample containing the trace metal or trace element

of interest will be aspirated as a spray in the chamber where it is
mixed with air and fuel. The mixture passes through the mixing
baffles where large drops are drained and only fine droplets will
reach the flame. The light coming from the light source will have
to pass through the ground-state atoms present in the sample.
The amount of light absorbed by the atoms present in the
sample is proportional to the concentration of the analyte of The light source strikes the beam of light into the
interest. A beam of light strikes now from the atoms present in primary filter. The mechanical attenuator control the light
 CLINICAL CHEMISTRY
LECTURE / FIRST SEMESTER
intensity coming from mercury. Primary filter select the individual scattering from the particle. Minimum light scatter occurs at 90º
wavelength of light. From the primary filter, a beam of light to the incident beam.
strikes the sample holder or cuvet and from the sample cell, 2. Mie Theory
another beam of light strikes the secondary filter. The secondary - If the wavelength of light is less than the particle
filter passes longer wavelength of fluorescent light to diameter (d > 0.1λ), then the light scatters forward.
photodetector. The electrical output of a photodetector is
proportional to the intensity of the fluorescent energy and will be 3. Rayleigh - Debye Theory
displayed by the read-out device. - If the wavelength of light is approximately the same as
the particle size, more light scatters in the forward direction than
Advantages and Disadvantages of Fluorometry in other directions.
Advantages:
- Specificity
- Sensitivity (1,000 times more sensitive than most
spectrophotometric methods)

Disadvantages:
- Sensitive to environmental changes, use of
contaminated chemicals or solvents, change of solvents,
temperature changes
- Quenching: decrease in fluorescence due to these
changes

Chemiluminescence
- is different from fluoroscence in that no excitation
radiation is required and no monochromators are needed
- emitted radiation is measured with a photomultiplier
tube and the signal is related to the concentration of analyte of
interest Turbidimetry
- chemiluminescence reactions are oxidation reactions - measurements are made with a spectrophotometer to
of luminol, acridinium esters, and dioxetanes characterized by a determine the concentration of particulate matter in a sample
rapid increase in intensity of emitted light followed by a gradual - determines the amount of light blocked by a
decay suspension of particles; reduction of light scattering
- Applications: It is used in microbiology analyzers,
Advantages and Disadvantages of Chemiluminescence coagulation analyzers, and is used to quantify protein
Advantages: concentration in biologic fluids such us urine and CSF.
- Subpicomolar detection limits, speed, ease of use and
simple instrumentation

Disadvantages:
- impurities can degrade sensitivity and specificity

Turbidimetry and Nephelometry

- Principle: Turbidimetry and Nephelometry are based
on the scattering of radiation by particles in suspension
- Applications: Measurement of antigen-antibody
reactions, prealbumin, and other serum proteins

Nephelometry
- is the measurement of the light scattered by a
particulate solution.

Three Types of Light Scattering The light source strikes a beam of light into the cuvet. A
1. Rayleigh Theory collimator or lens regulates the light coming from the light source
- if the wavelength (λ) of light is greater than the to the cuvet. From the cuvet, a photodetector detects the amount
diameter (d) of the particle (d < 0.1λ), the light scatter is of scattering at 90º and in the forward direction in nephelometry
symmetrical around the particle. Equal distribution of light
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LECTURE / FIRST SEMESTER
and the amount of light blocked by the particular solution in the
forward direction for turbidimetry

Laser Applications pCO2

- LASER: Light Amplification by Stimulated Emission of - a pH electrode within a plastic jacket (has sodium
Radiation bicarbonate buffer and gas-permeable membrane)
- laser light can provide polychromatic light - the hydrogen ion is measured by potentiometric
- based on the interaction of radiant energy with excited indicator system
atoms or molecules CO2 + H2O ⇌HCO3- + H+
- can serve as source of incident energy in a Coulometry
spectrometer or nephelometer - measures the quantity of electricity (in coulumbs)
- Applications: Hematology and Flow Cytometer needed to convert an analyte to a different oxidation state
Analyzers for the differential analysis of White Blood Cell - follow the Faraday’s Equation
- Application: to measure chloride ion in serum,
Electrochemistry plasma, CSF, and sweat samples
- involves measurement of the current or voltage
generated by the activity of specific ions Amperometry
- is the measurement of the current flow produced by an
Potentiometry oxidation-reduction reaction
- most widely used technique in clinical measurement - Application: to measure chloride ion in serum,
- measurement of potential (voltage) between two plasma, CSF, and sweat samples; pO2 electrode (gas-sensing
electrodes in a solution electrode used to measure the partial pressure of oxygen in the
 Reference Electrode blood) in blood gas analyzers
- electrode with a constant voltage - follows Amperometric technique
- Most widely used: Calomel and Silver / Silver Chloride
 Indicator Electrode Voltammetry
- measuring electrode - is a method in which a potential is applied to an
 Concentration of Ions electrochemical cell and the resulting current is measured
- difference between two electrodes - resulting current is related to analyte measured
- the result of calculation is related to the analyte - Anodic Stripping Voltammetry - used to measure
concentration heavy metals such as lead
- Nernst equation

Ion - Selective Electrode (ISE) Osmometry

- Flame Photometry is no longer used because of ISE - is the measurement of the osmolality of an aqueous
- is very sensitive and selective for the ion it measures solution such as serum, plasma, or urine.
- uses specialized membrane depending on the analyte Osmolality: measure of the number of dissolved particles in a
of interest fluid
- consists of a membrane separating a reference - Osmotically Active Particles - Glucose, Urea,
solution and a reference electrode from the solution to be Nitrogen, Sodium; when these particles are added in a solution,
analyzed the osmolality increases
- Two Types: - Effects of an increase osmolality / Colligative
1. Direct - does not require sample dilution Properties:
2. Indirect - the sample requires dilution before 1. Osmotic Pressure increases
analysis phase 2. Boiling Point increases
 Glass aluminum silicate - sodium 3. Freezing Point decreases
 Valinomycin gel - potassium 4. Vapor Pressure decreases
 Organic Liquid ion exchangers - calcium and lithium  Osmometer
 Gas electrodes - carbon dioxide and ammonia - is used to measure the concentration of solute
 Enzyme electrodes - urease and glucose oxidase particles in a solution
 Freezing Point Osmometer
pH Electrode - Freezing point depression is proportional to the
- used to measure hydrogen ion activity number of solute particles.
- Buffer: has known hydrogen ion concentration -mOsm/kg
- Internal Reference Electrode: Silver / Sliver Chloride
- External Reference Electrode: Calomel Electrode
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LECTURE / FIRST SEMESTER
4. Zwitterion
- both positively charged and negatively charged at the
same time with an overall neutral charge

Factors Affecting the Mobility of Particles

The mobility of the particle is affected by the following factors:
 Net charge of the particle
 Size and shape of the particle
 Strength of the electric field
 Chemical and Physical properties of the medium
 Electrophoretic temperature

Components of Electrophoresis
Consists of a chamber containing a stirring wire, a thermistor (a
temperature-sensing device) connected to a read-out device.
The inner chamber consists of a sample, rapidly super cold. The
sample is then agitated using a stirring wire to initiate freezing.
The freezing point is detected by the thermistor and the
osmolality of the sample is converted to units of milliosmoles/kg
of water.

Electrophoresis
- the separation of charged compounds based on their
electrical charge
1. Power Supply
- the process of separating the charged constituents of
2. Buffer
a sample by means of an electrical current
3. Support Medium
4. Sample
Electrophoretogram
5. Detecting System

Power Supply
- supplies constant current or voltage in the system
- this drives the molecules through the support medium
- driving force
- voltage depends on the ionic strength of buffer

Buffer
- used to provide ions that carry a current and to
maintain the pH at a relatively constant value
Types of Electrophoresis - ions that will enable the movement of current and the
1. Iontophoresis migration of particles
- migration of small ions - pH and ionic strength of the buffer affects the analyte
- sweat test for cystic fibrosis - a mixture of proton-donating and proton-accepting
substances that functions to maintain pH constant.
2. Zone Electrophoresis  Barbital (veronal) pH 8.6
- migration of charged macromolecules in a porous  Tris-boric EDTA pH 8.7
support medium
- DNA, proteins, lipoproteins pH
- pH acidic = binds H+ = ampholyte becomes postitively
Charged Particles charge, cation = migrates to the cathode (negatively-charged)
1. Amphoteric - pH basic = loses H+ = ampholyte becomes negatively
- a substance that can have a negative, zero or positive charge, anion = migrates to the anode (positively-charged)
charge depending on conditions
2. Anion Ionic Strength
- Negatively charged - Low = more charge will be carried = faster mobility
3. Cation - High = less charge will be carried = slower mobility
- Positively charged
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Support Medium Applications of Electrophoresis
- a network of interacting fibers or a polymer that is solid 1. DNA Fractionation
but traps large amount of solvent in pores or channel inside
- must not interact with the analyte
 Cellulose Acetate
 Agarose Gel
 Polyacrylamide Gel
- Electroendosmosis: support media affect the analyte

1. Cellulose Acetate
- Cellulose is acetylated to
form cellulose acetate by treating it
with acetic anhydride
- separates serum proteins 2. Isoenzyme Determination
into 5 bands: Albumin, α1, α2, β, γ 3. Protein Fractionation
globulin
- Isoelectric Focusing

2. Agarose Gel
- used as a purified fraction of agar
- it is neutral and thus, does not produce
electroendosmosis
- separates proteins into 10 - 15 bands

3. Polyacrilamide Gel
4. Lipid Fractionation
- separation of protein based on charge and molecular
size
Electroendosmosis
- separates serum proteins into 20 or more fractions
- the movement of buffer ions and solvent relative to the
- Isoenzyme determination
fixed support is called endosmosis or electroendosmosis
- forms ionic ground preventing the particles from
Sample
migrating into the support medium
- sample cannot be separated from one fraction to
another

Chromatography
- a physical technique that separates mixtures into
individual components
- used to separate complex mixtures on the basis of
Detecting System different physical interactions between the individual compounds
Electrophoretogram and the stationary phase of the system
- result of electrophoresis consisting of separated
strands of macromolecules Basic Component
1. Mobile Phase
Components: - carries the complex mixture (sample)
1. Direct Observation 2. Stationary Phase
2. Staining - through which mobile phase flows
- specific for one chemical group 3. Column
3. Radioactive Dye - holds the stationary phase
- Iodine-125 4. Eluate
4. UV Visualization - separated components
- is the simplest way to detect
5. Densitometer Mode of Separation
- a densitometer is a device that measures the degree 1. Adsorption
of darkness (the optical density) of a photographic or 2. Partition
semitransparent material or of a reflecting surface. 3. Steric Exclusion
4. Ion Exchange
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Classification of Chromatography Based on their Stationary High-Performance Liquid Chromatography
Phase
1. Planar Chromatography
- stationary phase is coated on a sheet of paper or
bound to glass or plastic plate
- Paper Chromatography

2. Column Chromatography
- the stationary phase is packed into a tube or coated  Pump
onto the inner surface of the tube/column - forces the mobile phase through the column
 Thin-layer chromatography  Columns
 Gas chromatography - holds the stationary phase
 Liquid chromatography  Sample Injector
- introduce the sample into the mobile phase
Planar Chromatography  Detectors
Paper Chromatography - produce an electronic signal proportional to the
- Fractionation of sugar and amino acid concentration of separated component
- Sorbent-Whatman paper - Photomultiplier Tube - most common photodetector
- The solvent and fractions move up at different rates in spectrophotometer
- Retention Time: Able to separate and identify  Recorders
components in a chromatographic eluate, by comparing and - Isocratic Elution: elution strength of the mobile phase
calculating is constant

Column Chromatography Mass Spectrophotometer

1. Thin-layer Chromatography - fragmentation and ionization of molecules using a
- Drug Screening suitable source of energy
- Semiquantitative screening test - before detection and quantification by Mass
- Stationary Phase: Thin layer sorbent coated on a Spectrophotometer, it must be separated by Gas
glass plate or a plastic sheet Chromatography
- The mobile phase is place in one edge of the plate - it can also detect structural information and
which migrates up the thin layer determination of molecular weight
- Each developed spot is measured by densitometer - an analytical technique in which chemical compounds
- Concentration is calculated by comparison with a are ionised into charged molecules and ratio of their mass to
reference standard charged measured (m/z)
- Eluate:  Quadrupole Mass Analyzers
 Ion Trap Analyzers
 Time of Flight Analyzers
- Ionization of samples:
 Electron Spray Ionization (ESI)
 Matrix Assisted Laser Desorption Ionization
- Time of Flight Procedure (MALDI - TOF)
Procedure:
1. Fragmentation of molecules using Gas Chromatography
2. Ionize the sample using ESI and MALDI
3. After exciting, measure the mass to charge ratio (which is
2. Gas Chromatography distinct to the molecule) using Quadrupole Mass Analyzers or
- Used to separate mixture of compounds that are Time of Flight Analyzers
volatile made or can be made volatile
- GCMS: Gas Chromatography Mass Spectrometry Automation
Automation
3. Liquid Chromatography - the process whereby an analytical instrument
- HPLC: High-Performance Liquid Chromatography performs many tests with only minimal involvement of analyst;
- Uses pressure for fast separation of thermolabile the controlled operation of an apparatus, process, or system by
substance mechanical or electronic devices without human intervention
- when exposed to high temperature, it becomes - mechanization of the steps in a procedure
unstable
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Batch Analysis 2. Simultaneous Multiple Analyzer (SMA)
- type of analysis in which many specimens are - SMA-6 and SMA-12
grouped in the same analytical session - multiple channels; can produce 6 or 12 test results at
- all samples are loaded at the same time and a single a rate of 360 or 720 tests per hour
test is conducted in each sample
3. Centrifugal Analyzer
Carryover - First centrifugal analyzer (1970) from NASA outer
- the transport of a quantity of analyte or reagent from space research
one specimen reaction into and contaminating a subsequent one - Dr. Norman Anderson developed a prototype in 1967
- Additive carryover: transfer of additive from one at the Oak Ridge National Laboratory
collection tube to another - had carryover problems and costly reagent waste

Discrete Analysis 4. Automatic Clinical Analyzer (ACA)

- type of analysis in which the sample is aspirated into - the first non-continuous flow, discrete analyzer as well
the sample probe and then is delivered, often with reagent, as the first instrument to have random-access capabilities (1970)
through the same orifice into a reaction cup or another container - any test can be performed in any sample in any
sequence
Parallel Analysis - unique features: plastic test packs, positive patient
- type of analysis in which all specimens are subjected identification, and infrequent calibration
to a series of analytical processes at the same time and in a - Single-channel: Capable of providing a single test
parallel fashion result on approximately 40 samples per hour
- more than 1 test is analyzed concurrently on a given
clinical specimen 5. Thin Film Analysis Technology (1976)

Random Access Analysis 6. Kodak Ektachem (Vitros) Analyzer (1978)

- the most common configuration of an automated - the first to use microsample volumes and reagents on
analyzer, in which analyses are performed on a collection of slides for dry chemistry analysis (utilizes filter paper with
specimens sequentially and each specimen is analyzed or a impregnate reagents)
different selection of tests. *wet analysis - liquid reagents
- any test can be performed in any sample in any
sequence 7. Discrete Analyzers (since 1980)
- Astra (now Synchron) analyzers (Beckman Coulter) -
Sequential Analysis (Ion Selective Electrons) ISEs
- type of analysis in which each specimen in a batch - Hitachi analyzers (Boehringer-Mannheim, now Roche
enters the analytical process one after another, and each result Diagnostics) - reusable reaction disks and fixed diode arrays for
or set of results emerges in the same order as the specimens spectral mapping
are entered - Paramax: no loner available; primary tube sampling
- multiple tests are analyzed one after another on a (original container tube as sample container)
given specimen
Vitros Analyzer and Slides:
Throughput
- the number of specimens processed by an analyzer
during a given period of time, or the rate at which an analytical
system processes specimens.

Workstation
- a clinical laboratory workstation dedicated to a defined
task contains appropriate laboratory instrumentation to carry out
that task

History of Automated Analyzers 8. Automated systems that are commonly used in CC

1. Technicon Autoanalyzer laboratory today:
- First automated analyzer (1957)  Aeroset and Architect analyzers (Abbott
- continuous flow, single-channel, sequential batch Diagnostics)
anaylzer; capable of providing a single test result on  Advia analyzers (Siemens)
approximately 40 samples per hour  Synchron analyzers (Beckman Coulter)
 Dimension analyzers (Siemens)
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LECTURE / FIRST SEMESTER
 Vitros Analyzers (Ortho-clinical Diagnostics / Basic Approaches to Automation
Dupont company) 1. Continuous Flow Analysis
 Several Roche analyzers - liquids (reagents, diluents, and samples) are pumped
through a system of continuous tubing
- samples are introduced in a sequential manner
- air bubbles serve as separating and cleaning media
- carryover problems and wasteful use of continuously
flowing reagents
- Simultaneous Multiple Analyzers (SMA): Chem 1

2. Centrifugal Analysis
- uses the force generated by centrifugation to transfer
and then contain liquids in separate cuvettes for measurement at
the perimeter of a spinning rotor
- run multiple samples, one test at a time, in a batch
(Batch Analysis)
- Cobas-Bio (Roche Diagnostics): IL Monarch

3. Discrete Analysis
- the separation of each sample and accompanying
reagents in a separate container
- analyzers have the capability of running multiple tests
one sample at a time or multiple samples one test at a time
- most popular and versatile analyzers
- Architect C8000 (Abbott Diagnostics); Unicel DXC
6001 (Beckman Coulter); Cobas 6000 (Roche Diagnostics);
Vitros 5.1 FS (Ortho Clinical Diagnostics)

Steps in Automated Analysis

1. Specimen Identification and Preparation
 Manual process in most laboratories
 by using robotics
 bypass the specimen preparation by using whole
Driving Forces Toward More Automation blood for analysis
1. Turnaround time (TAT) demands  by using PST (serves as sample container in
2. Specimen Integrity analyzer) and perform primary tube sampling,
3. Staff Shortages eliminates the need to perform specimen alliquoting
4. Economic Factors  by using a bar code label affixed to the primary
5. Less Maintenance collection tube, transfer to the loading zone of
6. Less calibration analyzer where the bar code is scanned and the
7. Less downtime information is stored in the computer’s memory
8. Faster start-up times
9. 24/7 uptime 2. Chemical Reaction
10. Throughput computer and software technology A. Specimen Measurement and Delivery
11. Primary tube sampling  most instruments use either circular carousels or
12. Increasing the number of different analytes on one system rectangular racks as specimen containers for holding
13. Increasing the number of different methods on one system disposable sample cups or sample tubes
14. Reducing laboratory errors  Vitros Analyzers- sample cup trays are quadrants
15. Number of specimens that hold 10 samples each in cups with conical
16. Types of fluids bottoms
17. Safety
18. Environmental concerns (I.e., biohazard risks )
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LECTURE / FIRST SEMESTER
- Manufacture the reagent in two stable
components that will be combined at the moment
of reaction
 Delivery of reagents
 Syringes driven by a stepping motor pipet the
reagents into reaction containers
 Piston-driven pumps - air displacement in the
positive displacement pipets
 Closed tube sampling  Pressurized reagent bottles connected by tubing
- no need to remove the closure or the lid of the to dispensing valves, the computer controls the
collection tube prior to analysis because the sample probe opening and closing of the valves
of the analyzer will automatically pierce the rubber stopper  Vitros analyzers use slides to contain their entire
to aspirate sample reagent chemistry system
 Discrete Analyzers  3 or more layers – spreading layer; one or more
- the probe automatically dips into each sample cup and central layers; indicator layer
aspirates portion of the sample  Spreading layer - accepts the sample
 Continuous Flow Analyzers  Central layers - can alter the alliquot
- when the sample probe rises from the cup, air is  Indicator layer - where analyte of interest
aspirated for a specified time to produce a bubble in may be quantified
between sample and reagent plugs of liquid  Scavenger layer - filters substance that ay
- air bubble serves as separating and cleaning media interfere with the reaction
- the probe descends into a container where was
solution (deionized water) is drawn into the probe and
through the system
 Carryover is a major concern
- except in vitros analyzers because they have a
unique dispensing system. The probosis (mouth of
sample probe) is pressed into the pipet tip nad once
connected to the probosis, it moves over the
specimen to aspirate the required volume of the
sample. After that, this will automatically move into the
dry reagent slide and automatically dispense the
required amount of the sample. The pipet tip will be
ejected into the discard bin. Each sample would use
one pipet tip and it will automatically be
C. Chemical Reaction Phase
discarded.
 consists of mixing, separation, incubation, and
reaction time
B. Reagent Systems and Delivery
 if the cuvettes are reusable, then wash stations are
 Reagents may be classified as liquid or dry systems
set up immediately after the read stations to clean and
 Forms of dry reagents:
dry these containers
 Bottled as lyophilized powder - reconstitution
prior to analysis
a) Mixing
 Dry chemistry slide – for the Vitros analyzer,
 Continuous flow analyzers – through the use of
containes the entire reagent chemistry system
coiled tubing, liquid rotates and tumbles on each loop
 Reagent handling varies
allowing now the mixing of the reagent and the sample
- Keep all reagents refrigerated until the moment
 Centrifugal analyzers – Start-stop action of the
of need, then preincubate them to reaction
reaction tray or bubbling of air though the sample, air
temperature or store
bubbles serve as mixing media
reagents in a
 VITROS Microslide technology – spreading layer
refrigerated
 Most automated wet chemistry analyzers – stirring
compartment on the
paddles that dip into the reaction container to stir
analyzer
sample and reagents
- Reagents in a dried,
tablet form is
b) Separation
reconstituted when
 undesirable constituents that will interfere with an
the test is to be run
analysis may need to be separated from the sample
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LECTURE / FIRST SEMESTER
before the other reagents are introduced into the
system (e.g. protein)
 Continuous flow analyzers – dialyzer using a fine-
pore cellophane membrane
 VITROS Microslide technology – spreading layer
 Discrete analyzers – no automated methodology to
separate interfering substances

c) Incubation
 Discrete or Continuous flow analyzers – Heating
bath
 VITROS Microslide technology – precondition
station that would bring the slides to 37 ℃ before
slides enter the incubator
d) Reaction Time *LIS - Laboratory Information System
 may depend on the rate of transport through the
system to the “read” station Total Laboratory Automation
 reaction rate is controlled by temperature - The idea of totally automating a clinical laboratory has
its roots in Japan, and the process was first tried in the early
3. Data Collection and Analysis 1980s.
A. Measurement Phase - Advantages of TLA include a decrease in labeling
 Most common – visible and UV spectrophotometry errors, reduced turnaround times, and a reduction in full-time
 Record optical readings set at desired wavelengths equivalents.
 Slide technology – reflectance spectrophotometry, - Major limitations of TLA include the need for
the sample is usually dispensed in the spreading layer substantial financial investment and increased floor space may
and mixes with the reagent in the reagent layer, and exceed 4,000 ft2 (Wilson, 2003); the need for highly technical
the indicator layer where the analyte of interest is personnel that could man the operation of the testing process.
measured and in this layer it has chromogen which is - 3 Phases of Testing:
a chemical or a substance that produced a colored  Preanalytic (Sample Processing)
end-product and the intensity of the color is directly - centrifugation
proportional to the analyte concentration - volume checks
- clot detection
B. Signal Processing and Data Handling - decapping tubes
 most automated instruments print the results in - tube labeling and alliquoting
reportable form
 the demographic-sample information is entered in the  Analytic (Chemical Analyses)
instrument’s computer together with the tests required - sample introduction and transport to the
(in sophisticated systems); then the sample holder
identification is printed with the test results - addition of reagent
 Bar code–labeled samples can be loaded directly - mixing of the sample and reagent
on the analyzer without the need to enter - incubation
identifying information manually. - calculations

 Postanalytic (Data Management)

- signal processing
- data handling
- process control
- all procedures are focused on data
management