Instrumentation and Automation

7/30/2019 Instrumentation and Automation

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The primary objective of instrumentation is to provide the clinician with the

best possible data to be of value to the patient. Thorough understanding of the

principles associated with the machines used is quite necessary for the operators in

order to have an easier time in performing maintenance procedures, calibration,

and in troubleshooting problems that may arise.

Automation in the clinical laboratory has been driven by the need to create

automated systems which are capable of reducing or eliminating manual tasks in

perform analytical procedures, and thus enhancing the reduction of errors.

AUTOMATION

3 stages in automation:

Pre-Analytical Stage

The pre-analytical stage is concerned with the handling of the sample or

specimen before processing. The two goals involved in this stage are to minimize

non-value added steps in the laboratory process and to increase available time for

value-added steps in order to produce better results.

Automated pre-analytical system attempts to provide the user with some of

the tasks necessary to prepare samples for testing, namely: pre-sorting,

centrifugation, volume checks, clot detection, decapping, secondary tube labeling,

aliquoting, and destination sorting in analyzer racks.

Analytical Stage

The analytical stage is primarily concerned with the processing of the sample

or specimen.

Tasks:

1. Introduction of the sample may be accomplished through the use of

peristaltic pumps and positive liquid displacement.

2. Addition of reagent reagent used must be handled, prepared and

stored properly. Automated analyzer can be classified based upon reagent.

Automated analyzers classified based upon the reagent used:

o Open reagent system - a system in which the reagents other than the

instrument manufacturers reagent can be used.

o Close reagent system - the operator can only used the manufacturers

reagent.


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3. Mixing

Mixing devices and techniques used:

o Magnetic stirring

o Rotating paddles

o Forceful dispensing

o Use of ultrasonic energy

o Use of vigorous lateral displacement

4. Incubation - uses an elongated cuvet path length and fluorocarbon oil

incubation bath to maximize result accuracy by enhancing absorbance value,

while using microvolume technology for samples and reagent (Bayer

Diagnostics) or uses a thermal electric module in the shape of a ring tomaintain a constant temperature for analysis (Beckman Coulter).

5. Detection

o Absorption spectroscopy - principal means of measurement in

automated analyzer.

o Reflectance photometry adapted to dry slide analysis and used in

chemistry laboratories.

o Fluorescent compounds - used for measurement of drugs, hormones,

and vitamins in several immunoassay analyzers.

POST ANALYTICAL STAGE

The post analytical stage plays its role after processing of the sample or

specimen. The instrument computer plays a major role in this stage as it represents

a means to accomplish several tasks, which include signal processing, data handling

and process control.

The processing of data by computers has allowed automation of nonisotopicimmunoassays, reflectance photometry and other nonlinear assays because

computer algorithms can transform nonlinear standard input signal into linear

calibration plots. Computers can also perform data correction, subtract blank

response, and monitor patients result against reference values.


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INSTRUMENTATION

PRINCIPLES USED:

SPECTROPHOTOMETRY

Spectrophotometry involves the measurement of the amount of light

absorbed by a solution and relating it to the concentration of the solution. It uses

Beer-Lambert Law (often referred to as the Beers law), which 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, as its

fundamental principle.

8 basic components:

1. Light source - light source provides light and must provide enough energy orpower to measure the analyte of interest.

Tungsten lamp or tungsten-halogen lamp used for wavelengths in the

visible region

Deuterium lamp used for wavelengths in the ultraviolet region

Silicon carbide - used for wavelengths in the infrared region

2. Entrance slit - excludes unwanted or stray light and prevents scattered light

from entering the system.

3. Monochromator - produces the light of specific wavelength from the light

source. It arranges the wavelength of light in such a way that

results to the wavelength required in the process.

Quartz prism separates white light into a continuous spectrum and uses

the principle of refraction.

Diffraction gratings bends light and forms wave front for ultraviolet and

near infrared spectrum. It uses the principle of diffraction.

Interference filter based on constructive interference of waves, utilizes

several mirrors, and uses the principle of reflection.

4. Exit slit - also known as bandpass act as the passage way of the filtered light

from the monochromator to the sample cell.


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5. Sample holder - also known as cuvet or analytical cell, maybe square or

round in shape and is used to hold samples and must be scratch-free so as

not to obtain erroneous results.

Soft glass cuvet for acidic solutions

Borosilicate cuvet for strong basic solution

Quartz or plastic cuvet for ultraviolet measurements

6. Photodetector - convert transmitted energy into an equivalent amount of

electrical energy.

7. Signal processor - a device that alters the signal and filters it to remove

unwanted components.

8. Readout device

Monitor

Ammeter

Galvanometer

Recorder

REFLECTOMETRY

Reflectometry makes use of a filter photometer (reflectometer), which

measures the quantity of light reflected by a liquid samples that has beendispensed onto a coarse solid support. Reflectometry is used in urine dipstick

analysis and dry slide chemical analysis.

2 types of reflectance:

Specular reflectance occurs of polished surface

Diffuse reflectance occurs on nonpolished surface

MOLECULAR LUMINESCENCE SPECTROSCOPY (FLUOROMETRY)

Fluorometry is based on the principle of luminescence in wherein an

exchange of energy occurs when compounds absorb electromagnetic radiation,

become excited and return to an energy level lower or equal to their original level.

Chemiluminescence is an example.

Basic components:

Light source


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Excitation (primary) monochromator

Cuvet

Emission (secondary) monochromator

Photodetector

NEPHELOMERTY AND TURBIDIMETRY

Both nephelometry and turbidimetry are based on the principle of scattering

of radiation by particles in suspension. Nephelometry, which is often used in the

measurement of antigen-antibody reaction, is the measurement of the light

scattered by a particulate solution whereas turbidimetry, commonly used in

coagulation analyzers, measurement of antibiotic sensitivities, and quantification of

protein concentration in body fluids, is the measurement of the reduction in light

transmission caused by particle formation.

REFRACTOMETRY

Refractometry is based on the principle of light refraction, which is the

bending of light. Refractometry is used in measuring protein concentration, specific

gravity of urine, and column effluent of high-performance liquid chromatography

analysis.

OSMOMETRY

Osmometry involves the measurement of the osmolality of an aqueous

solution and is based on the measuring changes in the colligative properties ofsolutions owing to variations in particle concentration.

FLOW CYTOMETRY

Flow cytometry is the measurement of multiple properties of cells suspended

in a moving fluid medium. It is used to count and sort cells, is the core component

of hematology cell counters, and is the technology used to differentiate white blood

cells.

ELECTROCHEMISTRY

Electrochemistry involves the measurement of the current or voltagegenerated by the activity of specific ions.

Potentiometry the measurement of voltage between two electrodes in a

solution

Coulometry the measurement of the quantity of electricity needed to

convert an analyte to a different oxidation state


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Voltametry - a method in which a potential is applied to an electrochemical

cell and the resulting current is measure

aniodic strippling voltametry - an electrochemical technique used in

measuring heavy metals

Amperometry - the measurement of the current flow produced by an

oxidation-reduction reaction.

CONDUCTANCE

Conductance is a measurement of the ability of a solution to carry an electric

current. This principle is used monitoring water purity, measuring analytes in blood.

It is also the principle used in the components of detectors used in high

performance liquid chromatography (HPLC) and gas chromatography (GC), cell

counters, and capillary electrophoresis.

IMPEDANCE

Electrical impedance, a measurement based on the change in electrical

resistance across an aperture when a particle in the liquid passes through this

aperture, is primarily used in enumerating leukocytes, erythrocytes, and platelets.

ELECTROPHORESIS AND DENSITOMETRY

Electrophoresis is the separation of charged compounds based on their

electrical charge, and in order to obtain a quantitative profile of the separated

fractions, the principle of densitometry is used, which is performed on the stained

support medium.

ISOELECTRIC FOCUSING

Isoelectric focusing is a technique which is performed similar to other

electrophoresis methods, the difference lies on the medium through which the

separating molecules migrate through. In isoelectric focusing, the separating

molecules migrate through a pH gradient. This principle is useful in measuring

serum acid phosphatase isoenzymes and in detecting oligoclonal immunoglobulin

bands in CSF and isoenzymes of creatine kinase and alkaline phosphatase in serum.

CHROMATOGRAPHY

Chromatography is a method of separation which is based on the different

interactions of the specimen compounds with the mobile phase and with the

stationary phase as the compounds travel through a support medium.

Gas chromatography - useful for volatile compounds

2 types of stationary phases commonly used in gas chromatography:


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Gas-solid chromatography- solid absorbent and uses the same

material for both the stationary and the support phase

Gas-liquid chromatography - uses liquid phases to coat the solid

support material

Liquid chromatography - for better separation of thermolabile compounds

High performance liquid chromatography - better method of liquid

separation over other forms due to a superior resolution, shorter analysis

time, and a greatly improves reproducibility

5 commonly used separation techniques in liquid chromatography:

Adsorption (liquid-solid) chromatography - compounds are adsorbed to a solid

support

Partition (liquid-liquid) chromatography - separates compounds based on

their partition between a liquid mobile phase and a liquid stationary phase

coated on a solid support

o Normal-phase liquid chromatography - uses a polar liquid stationary

phase

o Reverse-phase liquid chromatography - uses a nonpolar liquid

stationary phase

Ion-exchange chromatography - uses column packings that have charge-

bearing functional groups attached to a polymer matrix and uses the

mechanism of the exchange of sample ions and mobile phase ions with the

charged group of the stationary phase

Affinity chromatography - uses immobilized biochemical ligands as the

stationary phase to separate a few solutes from other unretained solutes

Size-exclusion chromatography - separates molecules according to the

difference in their sizes

MASS SPECTROMETRY

Mass spectrometry is based on fragmentation and ionization of molecules

using a suitable source of energy. The resulting fragment masses and their relative


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abundance yield a characteristic mass spectrum of the parent molecule. Mass

spectrometry typically involves the following major steps which include, conversion

of parent molecule into a stream of ion; separating the ions by mass-to-charge

ratio; counting the number of ions of each type or measuring current produced

when the ions strike a transducer.

SCINTILLATION COUNTER

A scintillation counter is an instrument that detects scintillations (flashes of

light that occur when gamma rays or charged particles interact with matter) using a

photomultiplier tube and counts the electrical impulses produced by the

scintillations. Scintillation counting is usually applied in radio immunoassays.

2 types of scintillation method:

Crystal scintillation used to detect gamma radiation

Liquid scintillation used to count radionuclides that emit beta particles

CAPILLARY ELECTROPHORESIS

Capillary electrophoresis is a separation technique that is said to be better

than conventional electrophoresis and high performance liquid chromatography due

to its short analytical time, resolving power, and microsample volumes. Capillary

electrophoresis is applied in the separation of serum proteins and haemoglobin.

NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

Nuclear magnetic resonance spectroscopy is a technique for determining the

surface of organic compounds. It is non-destructive, but requires a larger volume of

the sample compares to mass spectroscopy. Nuclear magnetic resonance is a

phenomenon that occurs when the nuclei of certain atoms are immersed in a static

magnetic field and exposed to a second oscillating magnetic field. Lipoprotein

particle measurement is the most popular application of nuclear magnetic

resonance spectroscopy.

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Instrumentation and Automation