clinical laboratory equipments
DESCRIPTION
A brief description to lab equipmentTRANSCRIPT
CLINICAL LABORATORY EQUIPMENTS
Mustafa YAMACLI 504061405
Supervisor Inci CILESIZ
26.12.2006
CONTENTS
PAGE CONTENTS 2
LIST OF FIGURES 3
1. INTRODUCTION 4
2. COLORIMETER 4
3. SPECTROPHOTOMETER 5
3.1 FLAME PHOTOMETERS 8
3.2 FLUOROMETRY 9
4. AUTO ANALYZERS 10
4.1 AUTOMATIC CLINICAL ANALYZER (ACA) 11
5. CHROMATOLOGY 13
5.1 GAS-LIQUID CHROMATOGRAPHS 13
6. ELECTROPHORESIS 16
7. HEMATOLOGY 17
7.1 ELECTRONIC DEVICES FOR
MEASURING BLOOD CHARACTERISTICS 18
REFERENCES 20
LIST OF FIGURES
PAGE
Figure 1 General view of calorimeter 5
Figure 2 Block diagram of spectrophotometer 6
Figure 3 General view of spectrophotometer 7
Figure 4 Block diagram of instruments for (a) flame emission and
(b) flame absorption 8
Figure 5 General view of flame photometer 8
Figure 6 Block diagram of a fluorometer
Figure 7 General view of fluorometer 9
Figure 8 The block diagram of Auto Analyzer 9
Figure 9 The functional block of the ACA 11
Figure 10 The basic components of a GLC 14
Figure 11 Example of a GLC recording for the analysis of blood levels of
phenobarbital (peak a) and phenytoin (peak c). Peak b corresponds
to the level of heptabarbital (the internal standard) 15
Figure 12 General view of Chromatography 15
Figure 13 In an electrophoresis system, charged molecules move through a
support medium because of forces exterted by an electric field 16
Figure 14 Examples of patterns of serum protein electrophoresis 17
Figure 15 A block diagram of a Coulter Model STKS 18
Figure 16 Coulter STKS aperture bath 20
1. INTRODUCTION
The clinical laboratory is responsible for analyzing patient specimens in order to provide
information to aid in the diagnosis of disease and evaluate the effectiveness of therapy. The
major sections of the clinical laboratory are the chemistry, hematology, microbiology
sections and the blood bank.
The chemistry section performs analyses on blood, urine, cerebrospinal fluid (CSF), and
other fluids to determine hoe much of various clinically important substances they contain.
Most applications of electronic instrumentation in the clinical laboratory take place in the
chemistry section. The hematology section performs determinations of the numbers of
characteristics of the formed elements in the blood (red blood cells, white blood cells and
platelets) as well as test of the function of physiological systems in the blood. Many of the
most frequently ordered of these tests have been automated on the Coulter Counter. The
microbiology section performs studies on various body tissues and fluids to determine
whether pathological microorganisms are present. The application of electronic
instrumentation for the blood bank is in its infancy. A few systems that automate the basic
classification of the type of the blood product (ABO grouping) are currently being developed.
2. COLORIMETER
A colorimeter is a device used to measure the absorbance of a specific solution. It allows the
absorbance of a solution at a particular wavelength of light to be determined. The most
common application of a colorimeter is to determine the concentration of a known solute.
Different chemical substances absorb different wavelengths of light. The concentration of a
solute is proportional to the absorbance.
Figure 1 General view of calorimeter
The optics filter in the colorimeter is used to select the wavelength of light which the solute
absorbs the most, in order to maximise accuracy. Colorimeters usually measure results in
percent transmission, percent absorption, or both.
3. SPECTROPHOTOMETER
Spectrophotometry is the basis for many of the instruments used in clinical chemistry. The
primary reasons for this are ease of measurement, satisfactory accuracy and precision, and
the suitability of spectrophotometric techniques to use in automated instruments.
Spectrophotometry is based on the fact that substances of clinical interest selectively absorb
or emit electromagnetic energy at different wavelengths. For most laboratory applications,
wavelengths in the range of the ultraviolet (200 to 400 nm), the visible (400 to 700 nm), or
the near infrared (700 to 800 nm) are used; the majority of the instruments operate in the
visible range.
A spectrophotometer consists of two instruments, namely a spectrometer for producing light
of any selected color (wavelength), and a photometer for measuring the intensity of light.
Figure 2 is a general block diagram for a spectrophotometer-type instrument. The source
supplies the radiant energy used to analyze the sample. The wavelength selector allows
energy in a limited wavelength band to pass through. The cuvette holds the sample to be
analyzed in the path of energy. The detector produces an electric output that is proportional
to the amount of energy it receives, and the readout device indicates the received energy or
some some function of it. The amount of light passing through the tube is measured by the
photometer. The photometer delivers a voltage signal to a display device, normally a
galvanometer. The signal changes as the amount of light absorbed by the liquid changes.
Figure 2 Block diagram of a spectrophotometer
If development of color is linked to the concentration of a substance in solution then that
concentration can be measured by determining the extent of absorption of light at the
appropriate wavelength. For example hemoglobin appears red because the hemoglobin
absorbs blue and green light rays much more effectively than red. The degree of absorbance
of blue or green light is proportional to the concentration of hemoglobin.
When monochromatic light (light of a specific wavelength) passes through a solution there is
usually a quantitative relationship (Beer's law) between the solute concentration and the
intensity of the transmitted light, that is,
where Io is the intensity of transmitted light using the pure solvent, I is the intensity of the
transmitted light when the colored compound is added, c is concentration of the colored
compound, l is the distance the light passes through the solution, and k is a constant. If the
light path l is a constant, as is the case with a spectrophotometer, Beer's law may be written,
where k is a new constant and T is the transmittance of the solution. There is a logarithmic
relationship between transmittance and the concentration of the colored compound. Thus,
The O.D. is directly proportional to the concentration of the colored compound. Most
spectrophotometers have a scale that reads both in O.D. (absorbance) units, which is a
logarithmic scale, and in % transmittance, which is an arithmetic scale. As suggested by the
above relationships, the absorbance scale is the most useful for colorimetric assays.
Figure 3 General view of spectrophotometer
3.1 FLAME PHOTOMETERS
Flame photometer is an optical device to measure the color intensity of substances, such as
sodium, potassium that have been aspirated into a flame. Flame photometers differ in three
important ways from the instruments have been already discussed. First, the power source
and the sample-holder function are combined in the flame. Second, in most applications of
flame photometry, the objective is measurement of the sample’s emission of light, that is
shown in Figure 4. (a), the sample, combined with a solvent, is drawn into a nebulizer that
converts the liquid into a fine aerosol that is injected into the flame, rather than its absorption
of light, that is based on the fact that the vast majority of atoms in flame absorb energy at a
characteristic wavelength. Third, flame photometers can determine only the concentrations of
pure metals.
Figure 4 Block diagram of instruments for (a) flame emission and (b) flame absorption
Figure 5 General view of flame photometer
3.2 FLUOROMETRY
Fluorometry is based on the fact that a number of molecules emit light in a characteristic
spectrum -the emission spectrum- immediately after absorbing radiant energy and being
raised to an excited state. Figure 6 shows a fluorometer block diagram. The primary filter
passes only wavelengths that excite the fluorescent molecule and the secondary filter blocks
all scattered excitation wavelengths and passes only the scattered fluorescent wavelengths.
The secondary filter and detector are at a right angle to the primary beam in order to avoid
direct transmission of the light source through the sample to the detector.
Figure 6 Block diagram of a fluorometer
Figure 7 General view of fluorometer
4. AUTO ANALYZERS
An auto analyzer sequentially measures blood chemistry through a series of steps of mixing,
reagent reaction and colorimetric measurements. The block diagram of Auto Analyzer is
shown Figure 8.
Figure 8 The block diagram of Auto Analyzer
The Auto Analyzer is consists of;
Sampler: The sampler aspirates samples, standards, wash solutions into the system.
Proportioning pump: It mixes samples with the reagents so that proper chemical color
reactions can take place, which are then read by the colorimeter.
Dialyzer: It separates interfacing substances from the sample by permitting selective passage
of sample components through a semi permeable membrane
Heating bath: The heating bath controls temperature (typically at 37 °C), as temp is critical
in color development
Colorimeter: It monitors the changes in optical density of the fluid stream flowing through a
tubular flow cell. Color intensities proportional to the substance concentrations are converted
to equivalent electrical voltages.
Recorder: The recorder displays the output information in a graphical form.
4.1 AUTOMATIC CLINICAL ANALYZER (ACA)
The DuPont Automatic Clinical Analyzer (ACA) differs from high-capacity instruments that
it is oriented toward flexibility rather than maximizing throughput. It performs
determinations in serial rather than parallel, but it can select any of 40 tests for each sample.
A functional block of the ACA is shown in Figure 9.
Figure 9 The functional block of the ACA
The ACA uses the unique concept of combining the sample with the reagents in the
analytical test pack (ATP). There is a different ATP for each determination. During operation
of the ACA, the ATP moves from station to station on a conveyer; any sequences of ATPs
can be selected. The time required to perform any of the ACA determinations is 7 min, and
there is a 37-s spacing between completions of determinations. This means that the ACA can
perform any of its determinations as STAT requests. This feature, which give the clinical
laboratory an important capability, results in a higher cost per test than that found in other
automated methods. The basic operations carried out by each subsystem are as follows:
Patient Identification: When the sample kit first enters the ACA, it passes through a station
where the patient –identification information that has been entered manually on the patient-
identification card on the side of the sample kit is transferred to the printer paper.
Filling Station: In the filling station, aliquots (measured liquid volumes) of the sample are
withdrawn from the sample kit and mixed with a diluent (which may be different for
different determinations). Then 5 ml of the combined solution is injected into each ATP. The
ATP’s binary code is read by electronic devices in the filling station to determine which
diluent to use for that particular ATP. After the ATPs have been filled, they continue to the
preheaters, and the sample kits are placed in the sample-kit exit tray.
Preheaters: In the preheaters, the ATP is heated to 37 °C; it is maintained at this
temperature for the remainder of the process.
Breaker-Mixer 1: At this station, the reagents in four of the plastic compartments are
crushed and mixed with the diluted sample.
Delay Stations: As the ATP successively passes through the five delay stations, the
chemical reactions of the first four reagents, the diluent, and the sample occur.
Breaker-Mixer 2: Here the last three reagent compartments are crushed and mixed with the
reaction solution. For some determinations, a delay is included here to allow sufficient time
for the reactions to go to completion before the ATP enters the photometer station. This delay
is controlled by the binary code that was read in the filling station and that identified the type
of test.
Photometer: In the photometer, the plastic envelope of the ATP is formed into a cuvette by a
unique pressure device. The pressure in the plastic pack is measured and used to determine
whether an adequate amount of sample and diluents has been inserted in the ATP. If the
pressure is too low, the test result is flagged with the letter P. the ATP binary code is again
decoded, and the photometer control board uses this information to select the measurement
method, filter type, and ADC constant.
Printer: The printer prepares the ACA report, which includes the patient-identification
information obtained in the filling station and the photometer results for all the ATPs filled
with the patient’s sample.
5. CHROMATOLOGY
Chromatography is a group of measurements for separating a mixture of substances into
components parts. The chromatograph utilizes an adsorptive medium, which when placed in
contact with a sample, adsorbs the various constituents of the sample at different rates. In this
manner, the components of a mixture are separated.
A chromatograph consists of a mobile phase, comprised of a solvent into which the sample is
injected – the solvent and sample flow through the column together - and stationary phase
where the material in the column for which the components to be separated have varying
affinities. The materials which comprise the mobile and stationary phases vary depending on
the general type of chromatographic process being performed.
5.1 GAS-LIQUID CHROMATOGRAPHS
The basic components of a Gas-Liquid Chromatographs (GLC) are shown in Figure 10. Prior
the being injected into the GLC, the patient sample usually must undergo some initial
purification, the extent of which depends on the determination that is being performed.
Figure 10 The basic components of a GLC
The activities step by step:
1. N2 or He carries and sweeps the sample and the solvent in which it travels through the
separation chamber (the column), this constitutes the mobile phase of the measurement.
2. Temp / pressure / pH are controlled in a particular sequence for maximal efficiency of
separation.
3. Introduces the sample into the column
4. The column is where the separation takes place. A glass or metal tube (1 m / ø7 mm) of
sufficient strength to withstand the pressures applied across it. The column contains the
stationary phase.
5. After the sample is flushed or displaced from the stationary phase, the different
components will elute from the column at different times. The components with the least
affinity for the stationary phase (the most weakly adsorbed) will elute first, while those with
the greatest affinity for the stationary phase (the most strongly adsorbed) will elute last.
6. A detector analyzes the emerging stream by measuring a property which is related to
concentration and characteristic of chemical composition. For example, the refractive index
or ultra-violet absorbance is measured.
Figure 11 shows a recording obtained from the analysis of a blood specimen for the levels of
the important anticonvulsant drugs Phenobarbital and phenytoin. A measured amount of
heptabarbital was added to specimen to serve as an internal standard. The area under the
Phenobarbital and Phenytoin peaks is compared with the area under the heptabarbital peak to
compute the blood levels of these drugs.
Figure 11 Example of a GLC recording for the analysis of blood levels of
phenobarbital (peak a) and phenytoin (peak c). Peak b corresponds to the level of
heptabarbital (the internal standard).
Figure 12 General view of Chromatography
6. ELECTROPHORESIS
Electrophoresis is an analytical method frequently used in molecular biology and medicine. It
is applied for the separation and characterization of proteins, nucleic acids and subcellular-
sized particles like viruses and small organelles. Its principle is that the charged particles of a
sample migrate in an applied electrical field. If conducted in solution, samples are separated
according to their surface net charge density, the most frequent applications, however, use
gels (polyacrylamide, agarose) as a support medium. The presence of such a matrix adds a
sieving effect so that particles can be characterized by both charge and size. Protein
electrophoresis is often performed in the presence of a charged detergent like sodium dodecyl
sulfate (SDS) which usually equalizes the surface charge and, therefore, allows for the
determination of protein sizes on a single gel. Additives are not necessary for nucleic acids
which have a similar surface charge irrespective of their size.
Characterizing samples by exploiting both differences in charge and size can yield much
more information. It requires that the same sample is analyzed not only in one, but several
gels. The procedure is more laborious; however the use of an automated electrophoresis
apparatus can make this a fast, routine procedure.
Figure 13 In an electrophoresis system, charged molecules move through a support
medium because of forces exerted by an electric field.
Figure 14 shows examples of the types of plots that are obtained via electrophoresis. These
plots are for serum protein electrophoresis.
Figure 14 Examples of patterns of serum protein electrophoresis. The left-hand
pattern is normal; the right-hand pattern is seen when there is an over production of a
single type of gamma globulin.
7. HEMATOLOGY
The blood consists of formed elements, substances in solution, and water. This section covers
only devices that measure characteristics of the formed elements: red blood cells (RBCs),
white blood cells (WBCs), and platelets. The primary functions of the RBCs are to carry
oxygen from the lungs to the various organs and to carry carbon dioxide back from these
organs to the lungs for excertion. The primary function of the WBCs is to help defend the
body against infections. Five types of WBCs are normally found in the peripheral blood. In
order of decreasing numbers in the blood of adults, they are neutrophils, lymphocytes,
monocytes, eosinophlis, and basophils . In disease, the total number and the relative
proportions of these types of WBCs can change; immature and malignant types of WBCs can
also appear. Platelets plug small breaks in the walls of the blood vessels and also participate
in the clotting mechanism.
7.1 ELECTRONIC DEVICES FOR MEASURING BLOOD
CHARACTERISTICS
There are two major classes of electronic devices for measuring blood characteristics. One
type is based on changes in the electric resistance of a solution when a formed blood element
passes through an aperture. The Coulter Corporation, Clay Adams, Lors & Lundberg, and
bker Diagnostics manufacture hematology instruments based on this technique. The other
type utilizes deflections of a light beam caused by the passage of formed blood elements to
make its measurements. Technician Corporation is a leading manufacturer of hematology
instruments that uses this approach. Coulter Corporation has been a leader in blood analyzers
for many years and it has developed a large series of instruments. Let us review the most
recent and most widely used instrument in this series, the Coulter STKS. The analyzed
sample is blood that has been anticoagulated, with ethylenediaminetetraacetic (EDTA).
Anticoagulants are substances that interfere with the normal clot-forming mechanism of the
blood. They keep the formed elements from clumping together, which would prevent them
from being counted accurately. EDTA does this by removing calcium from the blood. The
initial step in the analysis procedure is the automatic aspiration of a carefully measured
portion of the specimen. Next the specimen is diluted to 1:224 with a solution of
approximately the same osmolality as the plasma in Diluter I, Figure 15. The diluted
specimen is then split, part going to the mixing and lyzing chamber and part to Diluter II.
Figure 15 A block diagram of a Coulter Model STKS.
The function of the diluting and lyzing chamber is to prepare the specimen for the
measurement of its hemoglobin content and WBC count. The lyzing agent causes the cell
membranes of the RBCs to rupture and release their hemoglobin into the solution. The
WBCs are not lyzed by this agent. Adding the volume of lyzing agent increases the dilution
to 1:250. A second substance, Drabkin’s solution, is present; it converts hemoglobin to
cyanmethemoglobin. This is done to conform with the accepted standard method for
determining hemoglobin concentration. The advantage of this method is that it includes
essentially all forms of hemoglobin found in the blood. The specimen is next passed through
the WBC bath, which functions as a cuvette for the spectrophotometric determination of the
hemoglobin content. The final step in this process is measurement of the WBC count.
Figure 16 Coulter STKS aperture bath
Figure 16 outlines the method that is used in making this determination. The same method is
used for counting RBCs. A vacuum pump draws a carefully controlled volume of fluid from
the WBC-counting bath through the aperture. A constant current passes from the electrode in
the WBC-counting bath through the aperture to the second electrode in the aperture tube. As
each WBC passes through the aperture, it displaces a volume of the solution equal to its own
volume. The resistance of the WBC is much greater than that of the fluid, so a voltage pulse
is created in the circuit connecting the two electrodes. The magnitude of that voltage pulse is
related to the volume of the WBC.
REFERENCES
1. Medical Instrumentation –Application and Design-, John G. Webster, #rd edition,
1998
2. Encyclopedia of Chromatography, edited by Jack gazes, c2005
3. Medicinal Chemistry, Everdus J. Ariens, 1974
4. http://en.wikipedia.org/wiki/Spectrophotometer
5. http://www.humboldt.edu/~dp6/chem110/color/color.html