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UNIT: Basic Designs in Automation
6basic.wpd
Instrumentation IV
Task
To introduce basic designs and procedures in automation.
Objectives
Upon completion of this exercise, the student will:
1.
2.
3.
4.
5.
Discuss common clinical applications of non-photometric instruments.
Compare gamma radiation to electromagntic radiation.
Compare / contrast component parts of a scintillation counter and the spectrophotometer.
Discuss the principles, terms and applications of ion - selective electrodes.
Basic designs of the following automated chemistry analyzers:
a. DuPont ACA
c.
Abbott TDX
b. Beckman ASTRA d. Kodak Ektachem
Principle I
Laboratory Instrumentation: Clinical Applications B Non-photometric Equipment
Equipment
pH Meter
Clinical Applications
pH, Na+, K+
Reference electrode maintains
constant voltage (usually AgAgCl,
HgHg2Cl2 (calomel) in KCl solution.
Salt bridge completes electrical
circuit
Ion Selective Electrodes
Osmometry
a. freezing point
depression
b. vapor pressure (dew
pt)
Principle
Measuring Electrodes
pH
H+ sensitive glass for pH
Na+
Na+ sensitive glass for Na+
K+
Valinomycin absorbent for K+
Ca++
PO4 in matrix for Ca++
PCO2
PCO2 alters pH; H+ sensitive glass
PO2
PO2 B amperometric O2 reduced to
OHB (rate dependent on O2 conc.)
Measure osmolality indirectly by
measuring colligative property
paralleling osmotic pressure
osmolality
(osmometry)
Amperometric B Coulometric
Titration
Chloride
High voltage, silver electrodes
produce Ag+ (coulometric
generation). AgCl2 ppt. Excess
Ag+ signals endpoint.
Electrophoresis
protein, immunoglobulin,
hemoglobin, isoenzymes
Distance proteins migrate in a gel
when a current is applied depends
MLAB 2401 - Clinical Chemistry Lab Manual  B 41
B CPK
B LDH
on molecular weight and overall
charge of the protein.
Chromatography
CPK & LDH isoenzymes
and drug screens
Under specific conditions,
separation of mixtures depends
upon the relative amount of time
the specific compound is in a
moving state (liquid or gas) as
opposed to a stationary state of
crystal/solid.
Scintillation Counter
trace concentrations of
hormones and drugs
A particle tagged with a
radionuclide emits gamma rays
which strike a detector of a
scintillation counter producing an
electrical pulse of a size
proportional to the energy of the
gamma ray striking it.
Theory of Electrodes
The major advance in clinical chemistry was the development of the pH electrode and,
subsequently, other electrodes that make possible the selective measurement of particular ions
when they are immersed in solution mixtures. The instruments utilizing these electrodes measure
the potential difference that builds up at an interface when two different concentrations of the same
ion are in contact with each other. Electrodes are the detectors that are sensitive to this potential
difference.
When a metal is in contact with a solution of its ions, there is a tendency for the metal atoms to give
up electrons and to enter the solution. There is also a tendency for the metal ions in solution to take
up electrons and deposit as atoms on the metal surface. Charges build up near the interface
depending upon which reaction proceeds at the greater rate. When the first reaction proceeds
faster than the second one, the net reaction will be a layer of electrons forming a negative charge on
the metal surface and a layer of positive ions in the solution along the metal surface. An electrical
potential exists across the interface owing to the opposing charges on either side. When measuring
electrode is in circuit with a reference electrode, electrons flow through the system and produce an
electric current. If an opposing voltage is adjusted so that no current flows, the total potential
(voltage electromotive force, EMF) of the system is equal to the imposed voltage and may be
measured. This is the basic, greatly simplified principle of electrode measurements.
The interface may be solid-solution, or be one between two solutions containing the same ion in
different concentrations. In this case, the solutions are separated by a membrane that is permeable
to the ion. the potential across the membrane is dependent upon the relative rates of ion diffusion in
each direction through the membrane.
Each of these solution interfaces is called a half-cell. There must be two half-cells present in a
circuit for a potential difference to be measurable. Some half-cell potentials are extremely stable
and easy to reproduce. Since the potential of these half-cells can be established accurately, they
serve as reference potentials (or reference electrodes) against which unknown voltages are
measured. The reference electrodes consist of a metal and its salt in contact with a solution
containing the same anion. The two most widely used reference electrodes are the silver-silver
chloride electrode and the calomel electrode.
The calomel electrode contains mercury in contact with mercurous chloride (calomel) which is in
contact with a KCl solution. A small ceramic plug of fiber saturated with the KCl at the tip of the
glass envelope serves as a membrane and carries the current into the surrounding solution to
complete the circuit.
B 42  MLAB 2401 - Clinical Chemistry Lab Manual
UNIT: Basic Designs in Automation (continued)
The silver-silver chloride electrode consists of a silver wire coated with a deposit of silver chloride
immersed in a known KCl solution. As with the calomel electrode, the circuit is completed by a KCl
junction with the surrounding solution.
Frequently, the reference electrode is made quite small to enable it to be included in the glass
cylinder with the measuring electrode. This Acombination@ electrode is convenient and may be
used with much smaller volumes of solution than with two separate electrodes.
The indicator/measuring electrode's half-cell potential responds to the activity or concentration of the
species being measured. When a reference electrode and the appropriate measuring electrode are
immersed in a test solution and the circuit is completed, the voltage difference between them can be
measured. The potentiometer is calibrated against a solution of known ion concentration and the
unknown solution is then introduced. The measured voltage change is related to the change in ion
concentration. For H+, a change of 1 pH unit causes a voltage change of 59.15 millivolts at 25C.
Ion-Selective Electrodes B Electrodes have been developed for the potentiometric measurement
of many ions in addition to the hydrogen ion (pH) by the introduction of ion-selective membranes that
are sensitive only to certain ions. They operate in a fashion similar to the glass electrode in its
measurement of hydrogen ion concentrations. When an ion-specific membrane separates two
solutions that differ in concentration of that ion, a potential is developed across the membrane; the
size of the potential is dependent upon the difference in the ion concentrations. The membranes
vary widely in type, including selective glass membranes, membranes consisting of a crystal, an
immobilized precipitate, or a liquid layer.
The activity of any ion can be determined potentiometrically if an electrode can be developed that
responds selectively to the ion of interest.
1.
A sodium sensitive glass have been introduced that is insensitive to hydrogen ions and shows
a selectivity for sodium over potassium of about 300 to 1.
2.
A potassium electrode incorporating a valinomycin membrane shows a selectivity for
potassium over sodium of 1000 to 1.
3.
The development of an electrode selective for calcium ions has made possible the direct
measurement of ionized calcium, the physiologically active form.
4.
Chloride electrodes have long been used in the laboratory for the measurement of chloride in
sweat.
5.
Ion-selective electrodes have also been developed for many other ions, including lead, copper,
and ammonia.
6.
In addition, the use of selective electrodes in the clinical chemistry laboratory has grown by the
skillful combination of enzymatic action with selective electrodes. Such an instrument
(Beckman ASTRA) has been devised for the measurement of serum glucose, urea, and
cholesterol by utilizing specific oxidases to oxidize a substrate. Each reaction requires oxygen.
The instrument has a modified pO2 electrode to measure the rate of O2 utilization which is
proportional to the specific substrate concentration.
Beckman ASTRA
MLAB 2401 - Clinical Chemistry Lab Manual  B 43
UNIT: Basic Designs in Automation (continued)
Measurement Techniques B The ASTRA consists of a series of modules for each chemistry test.
Several of the modules utilize ion-selective electrodes.
A.
Enzymatic B Conductivity Rate Method B Measures the rate of increase in conductivity when a
known volume of sample is introduced into the reagent. The time rate of increase of solution
conductivity is directly proportional to the concentration.
Example: BUN
B.
Differential pH B Rate
Change
of
pH
B
Unknown sample introduced into the reaction cup and liberated gas diffuses through a
membrane lowering the pH of the solution. The rate of pH change is directly proportional to
the concentration of the gas producing substance. Second electrode serves as a reference.
Example: CO2
C.
Endpoint Titration B (Coulometric) B The sample is introduced into the reagent in a reaction
cup containing an electrode that is sensitive to the presence of specific ions in solution. The
number of ions required to titrate is directly proportional to the concentration of the unknown.
Example: Chloride
B 44  MLAB 2401 - Clinical Chemistry Lab Manual
UNIT: Basic Designs in Automation (continued)
D.
Timed-peak Rate of O2 Depletion B A sample is introduced into reagent in a reaction cup
containing an oxygen-sensitive electrode. The rate of oxygen consumption is directly
proportional to the concentration of analyte in the sample.
Example: Glucose
E.
Ion-Selective Electrodes B Sample is injected into buffer in a reaction cup where the
electrodes respond to an ion exchange that results in a potential change that allows for
calculation of analyte concentration when compared to a reference electrode.
Example: Sodium and Potassium
F.
Colorimetry/Conversion of NAD B NADH B A specimen is added to the reaction cup with a
specific reagent and the color-development measured. Procedures may be timed-rate, endpoint, turbidimetric, or kinetic.
Example: Creatinine (timed rate)
NOVA 4+4
The NOVA 4+4 in another example of an instrument that uses electrodes. It has a throughput of 80
samples/hour (320 tests/hour) and can be interrupted at any time to run a stat analysis. This
instrument will perform sample aspiration, analysis, clean-out and calibration, print and display
results, as well as monitor the performance of all system functions.
MLAB 2401 - Clinical Chemistry Lab Manual  B 45
UNIT: Basic Designs in Automation (continued)
Procedure II
Other Advanced Instrumentation
A.
DuPont ACA (discrete clinical analyzer)
1.
Measurement Techniques
a.
A sample is mixed with the contents of a reagent pack with the appropriate diluent
and a substance is produced which absorbs (or scatters) light.
b.
The amount of light absorption or the rate the substance is formed or converted is
proportional to the concentration or activity of the substance being analyzed (i.e.,
follows Beer's Law).
c.
Two absorbance measurements are made for each test and the difference between
the two (ΔA) are used to calculate the result using one of the following techniques:
1)
Two-filter Endpoint Chemistry Methods B In two-filter methods, the difference,
ΔA2, between the absorbance of the test solution at two wavelengths is
measured at the completion of the reaction. Example: Glucose is measured at
340 nm and 383 nm (single pack)
2)
Rate Chemistry Methods B In rate methods, the change in absorbance over
17.07 seconds, ΔA17 sec., is measured at one wavelength on a linear portion of
the reaction curve. Example: BUN is measured at 340 nm for 17.07 sec.
3)
Rate Enzyme Methods B The change in absorbance is measured over 17.07
sec., ΔA17 sec., at one wavelength on a linear portion of the reaction curve.
Indicator concentration is measured and related to enzyme activity and printed
in IU/volume. (1 IU - 1 umol per minute of substrate is converted to product
under optimized conditions.) Example: Assay for LDH
Lactate + NAD+
<
LDH
>
pyruvate + NADH (+ H+)
(non-absorbing at 340 nm)
4)
Two-pack Endpoint Chemistry Methods B The difference (ΔA2 packs) between
the absorbance of the measuring pack and a blank pack is determined at one
wavelength at the completion of the reaction. Example: Iron
Iron measuring pack
5)
(absorbing at 340 nm)
B Iron blank pack at 540 nm
Two-pack Enzyme Methods B The difference (ΔA2 pack) between the
absorbance of a measuring pack and a blank pack is determined at one
wavelength. Enzyme activity is calculated from the amount of product
produced by enzyme activity in 3.7 minutes. Example: Acid phosphatase
(ACP)
B 46  MLAB 2401 - Clinical Chemistry Lab Manual
UNIT: Basic Designs in Automation (continued)
thymolphthalein monophosphate
600 nm)
B.
ACP >
thymolphthalein + PO4(non-absorbing at
(absorbing at 600 nm)
Abbott TDX
The Abbott-TDX is an automated system for the quantitation of therapeutic drug
concentrations primarily:
1. antiarrhythmics
2. anticonvulsants
3. antineoplastics
4. antiasthmatics
5. cardiac glycosides
6. antibiotics
Sample analysis is based on the method of fluorescence polarization immunoassay.
Fluorescence labeled drug competes with the unlabeled patient drug for a limited number of
binding sites. This combines the principles of 1) competitive protein binding with 2)
fluorescence polarization providing both speed and specificity.
A sophisticated optical system detects a change in the polarization of fluorescent light emitted
by the dye, as tracer is bound. The concentration of drug in a sample can be extrapolated
from a calibration curve of polarization values vs. drug concentration. The tracer is a
fluorescein labeled drug that competes with unlabelled patient drug for a limited number of
specific antibody binding sites.
Measuring Fluorescence Polarization B The light source used for the fluorescence polarization
reaction comes from a tungsten halogen lamp. The light passes through a filter which selects
the correct excitation wavelength (485 nm). (A reference detector signal is used to monitor the
intensity of the lamp.) A liquid crystal-polarizer combination in the light path rapidly polarizes
the excitation beam horizontally and then vertically many times in sequence for each reaction
cuvette measured. The polarized excitation beam is focused with a culminating lens into the
center of the sample. Baffles bordering the cuvette serve as light traps preventing the
excitation beam from entering the emission optics. Another lens focuses emitted light and
passes it through an emission filter which selects light of a wavelength corresponding to the
emission peak of fluorescein (525-550 nm). Emitted light is then passed through a vertical
polarizer. A photomultiplier tube converts the fluorescence into an electrical current which is
recorded as numbers to be entered into the polarization equation. The polarization equation
stored in the computer memory is used to calculate polarization values for each reaction
cuvette measured.
Calculation of Concentration B The calibration curve for each drug assay is stored in
permanent memory in the TDX Analyzer. Concentrations of drugs in unknown samples are
read from this curve using the polarization values generated for each sample in the assay.
MLAB 2401 - Clinical Chemistry Lab Manual  B 47
UNIT: Basic Designs in Automation (continued)
C.
Kodak Ektachem
The Kodak Ektachem uses the principle of reflectance spectrophotometry where a beam of
light is directed at a flat surface and the reflected light is quantified. The surface to which the
light beam is directed, is a four layered slide containing dry reagents specific for one test
procedure.
After the test sample has interacted with the reagents, some light incident to the surface is
absorbed, and the remainder focused onto a photomultiplier tube. The term reflection density
is used to describe the absorption of light by chromophores at the surface. Reflection density
is related to the intensity of light reflected by the sample.
D.
Scintillation Counters
The scintillation counter is another laboratory instrument that measures electromagnetic radiation
emission. The scintillation counter measures gamma rays, rather than visible light.
Gamma rays come from an unstable nucleus that is rearranging to become more stable. During
this process they emit an energy in the form of gamma rays and/or particles. Gamma rays have
an extremely short wavelength and extremely high energy (high frequency). Unlike visible light,
they are usually discussed in terms of their energy rather than wavelength.
Because of their tremendous energy, instruments for measuring gamma rays must have a few
different components than the instruments which measure lower energy visible light. Gamma rays
are measured in a scintillation counter. (Beta emissions counters are also available.)
B 48  MLAB 2401 - Clinical Chemistry Lab Manual
UNIT: Basic Designs in Automation (continued)
Clinical chemistry procedures that use gamma / Beta emitters were very popular at one time due
to their high sensitivity in measuring very low levels of constituents such as drugs and hormones.
Today scintillation counters are rarely found outside of research institutions for two reasons: the
cost and hassle of nuclear waste disposal, and replacement of gamma - tags with enzymes.
MLAB 2401 - Clinical Chemistry Lab Manual  B 49
UNIT: Basic Designs in Automation (continued)
Name
Date
Study Questions
Instructions: Answer the following in the space provided. Unless otherwise instructed, each
question is worth one point.
1.
Basic electrolytes (Na+, K+, Cl-) and hydrogen ions are most frequently measured using what
methodology?
2.
List the non-photometric equipment commonly used for measuring the following. (2 point
each)
2.
a.
osmolality
b.
isoenzymes
List the predominant principle (i.e., photometry, electrodes, etc.) used in the majority of tests
measurements performed by the following. (1 point each)
a.
DuPont ACA B
b. Beckman ASTRA B
c. Abbott TDX B
3.
As you briefly outline the theory of fluorescence polarization immunoassay, state what two
principles are involved. (4 points)
4.
Outline the origin and pathway of light in the Abbott TDX. (2 point each, 3 points total)
5.
Define ion-selective electrode. (1 point)
B 50  MLAB 2401 - Clinical Chemistry Lab Manual
UNIT: Basic Designs in Automation (continued)
6.
Contrast reference and measuring electrodes. Provide an example of each. (3 points)
7.
Briefly explain the principle utilized by Kodak's Ektachem.
8.
What type of electromagnetic radiation is measured in the
scintillation counter?
9.
What is the source of gamma rays?
10.
If gamma / Beta emitting radioimmunoassay (RIA) procedures offered such
high specificity and sensitivity, why are they no longer widely used in the clinical laboratory? (2
points)
MLAB 2401 - Clinical Chemistry Lab Manual  B 51