Learning
Unit 7: SUPPLEMENT TO CHAPTER 6
DENSITOMETRY
This supplement to Chapter
6 will provide information on laboratory instrumentation that is used to
measure the density (concentration) of various protein matter that is deposited
on a support membrane following a procedure called electrophoresis. The instrument used to do this is a densitometer. The science of measuring density is called
densitometry. This supplement will help
answer the following questions:
1. What is
electrophoresis?
2. What is
densitometry?
3. What is a
densitometer?
4. What are
the principle components of a densitometer
5. What is the
clinical significance of densitometry ?
1)
What is electrophoresis?
Electrophoresis
is a technique used in most clinical laboratories. In electrophoresis, the impelling force is an electric potential
that tends to move electrically charged particles toward the anode or cathode,
depending upon their electrical charge.
Therefore, electrophoresis is an excellent separation technique. Electrophoresis has the ability to separate
rather large protein molecules from one another and deposit them on a support
membrane. This membrane can be enhanced
or treated with chemical solutions (stains) that react with the migrated
materials and form color complexes.
The
density of the color complex formed at a particular location on the membrane is
a direct indicator of the type and amount (concentration) of the matter. The following simple diagram illustrates the
principle of electrophoresis:
The
product of an electrophoresis is a support membrane that contains deposits of protein
material in direct proportion to the amount and type of proteins in the patient
sample. The example below shows these
deposits as dark concentrated areas called “bands”. Note that these “bands” vary in darkness or density. Research allows us to know what type of
protein migrates to each spot on the membrane.
We also then know if abnormally large or small bands occur, or if
irregular or unexpected bands appear on the pattern, that it is characteristic
of a particular disease. The
densitometer allows us to evaluate all of this information by transforming
these bands into meaningful clinical data.
2)
What is densitometry?
Densitometry
is the instrumental method of measuring the type and quantity of electrophoresis patterns on a support
membrane. The instrument used is a densitometer. In densitometry, the electrophoresis
membrane is moved past a measuring optical system and each band (sometimes
called a fraction) is detected, recorded, and integrated to provide
quantitative results. These results are
reported by type or class of protein as well as the concentration or quantity
of each band.
3)
What is a densitometer?
The
densitometer is an analytical instrument that optically detects and differentiates
electrophoresis patterns. The
accumulated data is electronically passed on to integrators that evaluate the
data and transform it into quantitative results. It is photometric system that usually measures visible or
ultraviolet light or both.0
Densitometers
can be stand-alone analytical instruments.
But, most laboratories today use systems that combine and integrate the
electrophoresis equipment and densitometry components into one unit. So when you hear the term “densitometer” it
can mean the instrument described in the paragraph above, but more likely, it
will describe the entire analytical system of electrophoresis and
densitometer. This combination proves
to be economical in that once the specimen is presented to the system, the
technologist can go on to do other tasks while the complete analysis occurs
without need for human intervention.
The
illustrations below show two types of modern systems:
On
the left is a semi-automated work center where the electrophoresis equipment is
on the lower right side of the instrument.
Following electrophoresis, the membrane is manually transferred to the
densitometer, which is on the lower left front of the instrument.
On
the right is an example of a completed contained and automated system. Once the specimens are placed on the
instrument, the system automatically performs the electrophoresis and then the
internal densitometer takes over and scans the electrophoretic pattern. The system is controlled by an onboard
microprocessor. The results are displayed
on a printout as well as a video display terminal. The onboard memory may store thousands of patient results.
4)
What are the principle
components of a densitometer?
·
Power Supply. The flow of current through a
membrane that offers resistance is associated with the production of heat.
Heat = (E)(I)(t)
Where
E = electromotive force
in volts
I = current in amperes
T = time in seconds.
The heat during the electrophoresis increases
the conductance of the system (decreases
the resistance).
Careful control of the heat produced is necessary. It is desirable to complete the
electrophoresis as quickly as possible.
But, heating increases the speed of the electrophoresis and too much
heat can cause the water in the support medium to evaporate. This causes further decrease the resistance
and the reaction goes out of control and yields meaningless results. To avoid this, a constant-current power
supply is necessary for controlling the electrophoresis to produce
meaningful results.
·
Timer. Exact timing of the electrophoresis portion
of the instrument is essential to produce accurate and precise results. Timing circuitry is fundamental to this
system
·
Incubator. Some systems require
temperature control outside the membrane surface during the electrophoresis,
staining and drying procedures.
·
Dryer. Basically a fan to help dry the
electrophoresis membranes in the warmed air provided by the incubator.
·
Scanning mechanism. A scanning mechanism is
needed to read (scan) the electrophoresis membrane or medium. Basically there are two types. One will move the optical system over the
stationary membrane or medium, the other will move the membrane or medium
through a stationary optical system.
Either way, the density is determined by the optical system.
·
Optical system. A system of producing all
light wavelengths is needed. Most
systems use tungsten bulbs to produce visible light. An additional light source is needed for ultraviolet light,
usually a deuterium lamp. Rarely, a
hydrogen lamp is used to provide the needed UV light. Most newer and automated systems provide both light sources. There are some models that provide only one
type of light source, either visible or UV.
The optical systems detect either absorbance or fluorescence. (See diagrams on the next page). Specific photodetectors transform the light into electrical energy. In some cases, there is need to amplify the detected energy. This must be routed through photomultiplier tubes and associated circuitry.
·
Optical system (continued) The electrical
signal that exits the photodetector is a mathematical function of the optical
density of the band on the electrophoresis membrane or medium. This information passes onto the next
component, the integrator.
IMPORTANT
NOTE: Recent advances in some
manufacturer’s optical systems eliminate the need for staining or drying
electrophoresis membranes or media.
While such devices will become prevalent someday, there will be many of
the older systems around for many years to come.
·
Integrators. The electronic signals from the photodetectors enable the
instrument to quantitate the protein materials detected during the
electrophoresis. With the aid of
computers, integrators analyze the electronic signals and transform the data to
either numerical data or graphical data.
With this information, the physician can determine whether the protein
patterns are normal or indicative of some disease condition.
5. What is the clinical significance of densitometry?
The question is perhaps better phrased “What is the clinical significance of electrophoresis?” Densitometry allows us to visualize the vast amount of clinical information yielded by electrophoresis.
Simply stated, electrophoresis is one of the two or three most powerful tools in clinical laboratory medicine. The human body is made up of chemicals---water, some elemental chemicals, and mostly molecules made up of protein materials. Some of these proteins are large (macromolecules) while some are extremely small (microproteins). All of the protein throughout the body is mixed together in our body fluids. Electrophoresis is the technique that allows us to separate these millions of proteins into meaningful information. It is truly a remarkable technique.
The clinical significance is difficult to explain because the applications are so vast and the information is limitless. Disease states are reflected by imbalances in the “normal” protein patterns. Some of these abnormal patterns are well known and are diagnostic of certain diseases. Some can only suggest a diagnosis and other analytical tools are needed.
Our genes are made of proteins. Perhaps the most exciting application of this laboratory technique in is the genetic field of DNA analysis. More discoveries are made each day that gain insight into the complexities of the effects our genetic material has on our health. Electrophoresis and densitometry are indispensable tools in genetics.
References:
1) Lee, LW and Schmidt, LM. Elementary Principles of Laboratory Instruments. 5th and 6th editions. 1978 and 1994.
2) Tietz, NW, Epstein, E. Textbook of Clinical Chemistry, pp. 98-110, 2nd edition. 1987.
3) WebMD, An internet service (adam.com) 2000
4) Technical Bulletin: Helena “Cardio REP System ; via Internet (www.helena.com)
5) Technical Bulletin: Beckman Coulter Paragon CZE 2000. via Internet www.beckmancoulter.com/beckman/clindiag/prodinfo/electrophoresis
Study
Questions & Answers
1) Electrophoresis is a laboratory technique that :
a) Concentrates chemicals for spectral analysis.
B Is an excellent way to separate protein molecules.
b) Measures pH
c) Differentiates between atoms and molecules.
2) In a clinical laboratory, a densitometer is:
a) A device that measures the thickness of cell walls.
b) An instrument that measures the amount of vitamins in blood.
c) An analyzer that optically detects and differentiates electrophoresis patterns.
d) A device used to measure mass and volume.
3)
What
property of matter must be carefully controlled by the power supply to enable
accurate and precise electrophoresis to occur?
a)
Heat.
b)
Melting
point.
c)
Mass
d)
Volatility
4)
Name
the two most common light sources used in modern densitometers.
a)
______________________________ Tungsten lamp (visible)
b)
______________________________ (Deuterium lamp) (UV)[Hydrogen OK]
5)
What
class of molecule does electrophoresis and densitometry typically analyze in
the clinical laboratory?
a) Protein
b)
Lipid
c)
Carbohydrate
d)
Drugs
Study
Questions
1) Electrophoresis is a laboratory technique that :
A) Concentrates chemicals for spectral analysis.
B) Is an excellent way to separate protein molecules.
C) Measures pH
D) Differentiates between atoms and molecules.
2) In a clinical laboratory, a densitometer is:
A) A device that measures the thickness of cell walls.
B) An instrument that measures the amount of vitamins in blood.
C) An analyzer that optically detects and differentiates electrophoresis patterns.
D) A device used to measure mass and volume.
3)
What
property of matter must be carefully controlled by the power supply to enable
accurate and precise electrophoresis to occur?
A) Heat.
B) Melting
point.
C)
Mass
D)
Volatility
4)
Name
the two most common light sources used in modern densitometers.
A)
______________________________
B)
______________________________
5)
What
class of molecule does electrophoresis and densitometry typically analyze in
the clinical laboratory?
B)
Lipid
C)
Carbohydrate
D)
Drugs