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