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The following can serve as guidelines in taking the eorts out of the process of computerization:
• Identify tasks that are best suited for the computer in the laboratory.
• Evaluate pre-packaged soware available for performing these functions.
• Investigate available computer models that can run the packaged soware per its speci-
cations, keeping in mind that expandability and exibility in the system are important (both in terms of data storage and future applications).
• Prepare laboratory personnel for the new technology, and reducing anxiety or ‘techno-
phobia’. The sta should be assured of the productivity gains to be achieved with the new machinery.
• Arrange for appropriate end-user technical training, diagnostic training as well as repair
assistance procedures.
• Setup backup procedures (preferably in manual form) for anticipated problems (lost
data, machinery failure, power failure, etc.) well in advance.
• Create backups oen, and make it a part of the prescriptive steps so that backup copies
of data les are always available.
• Assign a resource to champion the computer operations and function as the laboratory’s
on-site sta to run initial set of diagnostics in the event of a malfunction.
• Periodically repeat the aforementioned tasks of computerization upkeep, revise the
operational guidelines and retrain the sta on any new extensions or soware, as needed to ensure a smooth and uninterrupted operation in the laboratory.
Medical Laboratory Technology: Volume 3
automatIon In tHe cLInIcaL LaboratorIes of deveLoPIng countrIes
The rst automated system was introduced into the market by Technicon under the name of AutoAnalyzer. The company is now owned by Bayer. It is a continuous ow system where the specimens ow in a continuous stream while separated by air bubbles. At the nal stage, the air bubbles are released and colour of the mixture is measured by absorption photometry. Acid, phosphatases and bicarbonate—any of these can be chosen according to the need of the laboratory.
Centrifugal fast analyser is the next generation of automation and has many unique features. It is a discrete system where reagents and specimens are placed in the innermost discrete compartments in a rotor using positive displacement syringes. The reagents and samples get mixed and delivered to outer compartment by centrifugal force. The outer chamber provides appropriate reaction conditions and read the change in optical density at quick sequence each time the cuvee passes over the optical device during rotation. The instrument is very useful for the determinations in rate reactions.
The dry slide technology has become quite popular not only in large laboratories but also serve the needs of POC in the physician’s oce. The colour reactions in dry slide technology are read through reectance photometry.
Dade Behring (formerly Dupont) is marketing a dierent kind of discrete analyser. All the reagents are put in dierent pouches of a plastic bag where they are mixed with the specimen and the mixture undergo the required chemical reaction conditions. Finally, the optical absorption of the reaction uid is read at specic wavelength through the plastic bag. A bar code marked at the top of the bag prompts the machine to respond to specic test.
We will focus here on three automated analysers which are commonly seen in various laboratories of India and other developing countries—AutoAnalyzer (Bayer), Clinical Corona (Boehringer Mannheim) and Auto Pacer (Miles of India Ltd.). We will focus here the operation of these analyzers.
Automation in Clinical Biochemistry
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AutoAnalyzers
This is grouped under the ‘continuous ow’ where the specimens move in a stream, separated by air bubbles. In discrete system all specimens are independent of each other and one can have multiple choice of tests and they can have random access. Three unique approaches of the discrete system include centrifugal fast analysers, dry slide technology and compartmental pre-packaged reagents that move on a belt, meeting all the reaction conditions and ultimately subjected to optical measurements before packages are disposed o. Bulk reagents are available and spare parts are becoming easier to obtain through the Internet.
Components of AutoAnalyzer
The ve major components of the AutoAnalyzer are shown in Figure 32.3.
Figure 32.3 Continuous ow automated system. Components and working of AutoAnalyzer for the
analysis of blood urea nitrogen (BUN) by diacetyl monoxime method.
Sampler and Cam The sampler is a circular platform that holds the cups containing the standards and specimens for analysis. As the sampler rotates, it brings each cup in turn under the sampling probe, which aspirates for a FT, decides sample volume, and alternates with the wash cycle. The dwell time in the specimen and the sample-to-wash ratio are governed by the cam. An appropriate cam is selected to determine the rate of analysis. The most common sample-to-wash ratio is 2 : 1.
Pumps and Manifolds The pump is the heart of the AutoAnalyzer, and the manifolds are the arteries. The proportionating pump has a peristaltic action that moves the uids inside the tube in one direction at a constant speed, rendering a uniform rate of delivery throughout the system. The advancing movement accomplishes sample aspiration reagent pickup, mixing, and all other actions. The diameter of the manifold tube determines the volume of the uid. Before the sample stream enters the dialyser, air bubbles are introduced in both specimen and reagent streams. The air bubbles help in cleaning the manifold tubing and avoid carry­over eects. In an enzyme study, substrate and the serum specimen (containing enzymes) are rst mixed in an incubation chamber kept at 37°C for a certain period. The extended coil determines the time delay. Aer incubation, the specimen stream with products of the substrate enters the upper chamber of the dialyser.
Dialyser The dialyser is a double compartment separated by a semi-permeable membrane. A diluted sample stream (‘donor stream’) circulates on one side of the membrane while the recipient stream (generally one of the reagents or saline solution) circulates through the other side. The membrane allows part of the sample constituents of low-molecular mass (the analyte) to pass through while it holds back, the compounds with high-molecular mass (protein) that ow into the waste. The amount of solute that passes through the membrane is
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inuenced by the instrumental factors, which are kept constant and the variable concentrations gradient, which is the basis of ow analysis. Although only a fraction of the total amount of the analyte present in a unit volume of specimen passes through the membrane, the ratio of diusion remains constant. The AutoAnalyzer operates on the accurate measurement of the sample-standard ratio. It is not important to have the total amount of compound to be taken for analysis, nor is it necessary to take the chemical reaction to completion. As long as a photometric measurement is possible at the nal step, there is no loss of accuracy.
Reaction Chamber or Heating Bath This provides elevated temperature and a time delay which are required for the development of a coloured reaction product. The temperature is usually maintained at about 95°C, and occasionally at 37°C, depending on the analysis. The time delay is accomplished by introducing a long glass coil inside the chamber in continua­tion with the manifold tubing.
Detector and Recorder The basic analytic procedure of the AutoAnalyzer is colorimetry. Thus the colour of the reagent stream, following the chemical reaction, is proportional to the amount of reacting compound. A small amount of coloured reagent is drawn by the continuous ow system into a microcuvee. The air bubbles are discarded by the ‘F’ tube or debubbler. To avoid uctuation of the light source caused by voltage uctuation, the colorimeter employs a dual-beam system. Light from a single tungsten lament lamp is split and collimated into two beams, one of which acts as a reference (null balance) while the other goes through the cuvee. Initial baseline conditions are achieved by controlling the light intensity of the reference beam through the introduction of a suitable aperture plate. The photocell of the colorimeter reads the light energy that is transmied through the coloured solution (%T) and converts it to electrical energy. The electrical impulse is nally communicated to the mechanical device of the recorder or directly to the computer.
The recorder chart provides a continuous measurement of the intensity of the light that has passed through the ow cell and is received by the detector (%7). The response is shown as peaks indicating the concentrations of the analytes. The highest concentration is at the centre of the peak with adjoining slopes that indicate a decreasing concentration of the analyte in the wash cycle phase. The peaks obtained from the specimen on the recorder chart are compared against the peaks of the standards. It is therefore important to calibrate the AutoAnalyzer prior to the running of specimens. Variation from a smooth shape of the peak frequently indicates many internal problems. For example, a sharp spike in the middle of a peak or between peaks indicates the presence of air bubbles at the time of colour measurement; trailing indicates possible obstruction in continuous ow; and overlapping peaks are indicative of poor wash.
Medical Laboratory Technology: Volume 3
Clinical Corona
The Clinical Corona (or Corona) made by Boehringer Mannheim is a discrete, compact and fully automated clinical batch analyser (Figure 32.4). It is a bench-top model that requires minimal space. A microcomputer system controls the whole analytical process via its programs. The program to be used in a specic assay is’ selected by the operator, who enters an analysis code through the keyboard.
Corona performs methods for routine biochemistry and is also capable of handling special tests with appropriate reagents. Its throughput (maximum number of tests that can be performed per hour) is 200 samples per hour for end point analysis (single observation) and 140 samples per hour in kinetic mode (multiple; observations) for enzyme assays. The required serum volume for a set of 20 chemistries is as small as 500 mL, and the reagent consumption per test is typically 400 mL (Figure 32.5).
Substances analysed (kinetic or end point) in the Corona are: Glucose (hexokinase), total protein (biuret), albumin (bromocresol green), bilirubin (dichloro diazophenyl), creati­nine (Jae), triglyceride (ATP method), urea (UV method), uric acid (UV method), calcium (o-cresolphthalein complexone), cholesterol (p-aminophenazone) and iron (bathophenan­trolin). Other tests can be introduced according to the need of the laboratory.
The enzyme assays (kinetic) include—SCOT, SGPT, LD, CK, ALP and GGT.
Automation in Clinical Biochemistry
Figure 32.4 Use of microprocessor in the operation of laboratory instruments. The communication
between different modules in the operation of Clinicon (Boehringer Mannheim, Sweden).
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Components of Corona
Various components of Corona and their functions are described here (Figure 32.5). It is important that the operator understands the function of each component in order to trouble shoot.
Sampler The sampler is a 40-position carousel that carries the specimen. Specimens must have clear identication, and the sequence must be noted in a register. Movement of the sampler is controlled by the microcomputer. The slots are numbered and it is possible to run replicates and standards simultaneously by choosing the slots.
Diluter This prepares the specimen for the assay, mixes the reagent and the sample, and delivers it into the reaction chamber. It is fully integrated to function with a single syringe and one valve whose movements are totally controlled by the microcomputer. The diluter has a small dead volume as only one syringe is involved in the whole dilution process. The dilution cycle begins by sucking the reagent into the syringe. The valve is switched on and thereaer the sample is aspirated and separated from the reagent by air. When the pipee has le the sample cup, another segment of air is sucked in and the outside of the pipee is wiped in order to minimize carry-over. The pipee is then transferred to the cuvee into which the sample and reagent are delivered. The volumes of the uids are regulated by the length of the stroke, and pickup deliveries of the uids are regulated by the valve. The syringe is capable of delivering 5–1000 mL of the specimen.
Reaction Chamber The main chemical reaction for the test is in the reaction chamber which is temperature controlled and newer chemicals are added. The instrument uses disposable cuvees (which add to the cost of its running) which are placed in racks in the in-feed area of the instrument, and end up in the out-feed area when the analytical process is complete. Just before entering the thermostat the cuvee receives the reagent and sample previously picked up by the diluter from a cup in the sampler. The racks travel through the thermostat and during this travel the reaction mixture is gradually heated to the set temperature. At any point in time during this feeding through the thermostat it is possible to add two additional
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Medical Laboratory Technology: Volume 3
Figure 32.5 Discrete automated system—Corona (Boehringer Mannheim, Sweden): (a) Principle
components of Corona; (b) Operation of diluter: (1) Filling of reagent, (2) Aspiration of specimen, and (3) Transfer and delivery of specimen and reager; (c) Filter disc used in the photometric system (visible range)
reagents. These reagents are preheated in order not to upset the temperature of the reaction mixture already in the cuvee. The pipee, tubing and heating block are in one piece which is easy to position and remove from the top of the thermostat. The reagent pumps are controlled by the microcomputer.
Detector The detector is built-in within the reaction chamber. The detector is an optical system which is typical of any photometer. Contents of the cuvees are well mixed before taking optical measurements. Most observations are made in the visible range, using a tungsten lamp but the instrument is capable of working in the ultraviolet range as well. Radiant energy from the light source passes through two lenses and the emerging parallel beams of light then enter the interference lter in order to choose the desired wavelength of light to go through the cuvee with the test solution. The parallel beam of light is then focussed by another lens in order to concentrate it at the middle of the cuvee. The light is absorbed by the test solution and the non-absorbed light is focussed by another series of lenses onto a silicon photodetector. The electrical signal sent by the photodetector is amplied, converted into logarithmic and digital form, and nally transferred to the microprocessor.
Automation in Clinical Biochemistry
Printer Aer receiving the signal from the CPU the microprocessor then performs the necessary calculations and transfers the nal information to the output printer.
Microcomputer System The microcomputer is the brain of the machine and controls the whole instrument through the CPU. The microprocessor receives instructions from the so­ware (program) which is operated by the user of the machine. The memory of the computer has two components, the user le (comparable with random access memory, RAM) and the master le which is provided by the manufacturer. The operator, by a few commands via the keyboard can make a selection of 16 analyses which are automatically loaded into the user le. These analyses are immediately accessible for routine use. Only one analysis should be run for a batch. For dierent analyses, dierent chemicals and reaction conditions will be needed.
There are three assay modes—constant rate (CR), xed time (FT) and end point (EP). Depending on the chosen mode, the arithmetic-logic unit of the CPU makes the necessary mathematical calculations. CR mode is used for enzymes; the slope is determined by the best t to measured data. The accuracy is checked and compared to a pre-set value (RMS) to yield a factor. The concentration results are obtained by multiplication by the factor. Fixed time mode is used for substrates and immunoglobulins. The primary value is determined by integration of the measured data forming the midpoint (t/2) of the reaction rate until the plateaux is reached. The EP mode is applied for routine chemistry—albumin, bilirubin, calcium and others. In the EP mode, the absorbance level is determined by the best t to measured data, and the concentration result is obtained by comparison with standards.
Before starting the machine load the carousel with specimens, check the identications of the specimens, place the standards, record the sequence, orime the diluter with the reagent, check the temperature light (it must be on), place the empty cuvees in position, and enter the analysis code, date and sequence number on the keyboard. Then press the ‘start’ key provided on the keyboard.
The Corona is programmed to check the QC. If a result does not full a pre-set QC parameter or unit value the result will be accompanied by a text or a symbol which depends on the type of error. Error messages are also given when the standard curve of an assay is unacceptable.
When the analysis code is entered by the user, all the seings are completed. The Clinicon Corona, however, is exible enough to adapt to newer methods to meet the needs of the individual laboratory. In order to adapt to newer methods, 38 parameters have to be xed such as temperature, wavelength, concentration unit, etc.
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Auto Pacer
The Auto Pacer (Chemetrics Analyser–I) made by Ames, USA (Miles of India Ltd.) is a computerized discrete analyser of modular design. It is a bench top model which is fully automated right from the sample dispensing to the nal printout of the results. The technician has to load the machine with samples, provide appropriate reagents, instruct the computer through the computer keyboard and set the parameters as required by the tests. It is capable of doing 26 biochemical tests. Although the analyser is pre-programmed for 26 tests, 37 programs are available and it is open for the addition of new tests as they are developed in future. There are also two open programs available to the user (one kinetic for enzyme study and one EP for routine chemistry). Over and above all these programs, a built-in statistical program for QC, i.e., for calculating standard deviations, coecients of variation, and means, is also available. It is faster than Clinicon Corona and can handle 300 EP reactions per hour (throughput) and 40 kinetic enzyme assays per hour. The analyser processes the sample in batches and it is possible to interrupt a batch run to do a ‘Stat’ test. The reagents are available from the manufacturer and can also be prepared in the laboratory.
The program provided by the manufacturer is not totally inaccessible. The user can change the test parameters, if needed, such as the number of data points, range of normal and others. The instrument is also designed for handling enzyme immunoassays, and has proved to be an important tool in drug analysis.
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Medical Laboratory Technology: Volume 3
Components of Auto Pacer
The modular design provides the following components (Figure 32.6):
Figure 32.6 Various components of Auto Pacer: (a) Digital display and printer, (b) Specimen pickup
module, (c) Specimen and reagent pickup syringes, and (d) Photometric module
Sampler It is a turntable carrying 60 reaction cups and 60 sample cups per tray. The carousel can be separately loaded with fresh specimens while the tests are in progress with one batch.
Automatic Dispenser This component functions in the automatic dispensing and diluting of sample and reagents. Pump and syringes are housed in this module.
Detector This consists of a spectrophotometer for photometric measurements. The spectro­photometer is of high precision and narrowband (8 nm) that incorporates a digital readout. It works in the entire range of photometric determinations for routine biochemical analyses (335–850 nm).
Computer This is the brain of the machine. The keyboard is the communication link between the user, the manufacturer ’s soware and the microprocessor. The temperature of the reaction chamber and ow cell is controlled by an internal thermostat (25–37°C). It is aached to the instrument and is programmed to function according to the test requirement. It has a built-in printer that gives impact paper printouts. If the results are not normal, the printout gives an automatic ag of abnormal, non-linear, invalid or out of range values.
The instrument is capable of performing EP, kinetic or initial rate reactions as the need be. It is designed for continuous batch operation round the clock.
SEAC (Ames)
This is a semi-automated analyzer (Ames, USA; Miles of India Ltd.) where the technician and the instrument works like a sophisticated spectrophotometer do the initial preparations. The
Automation in Clinical Biochemistry
advantage of this machine is that the use of separate cuvees is avoided, and the conditions of the test can be programmed. Boehringer Mannheim and other manufacturers have also marketed similar products. This instrument is highly eective in measuring enzyme kinetics and is a necessary tool for enzyme-linked immunoassays.
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PoInt-of-care testIng: a new aPProacH
In recent years, there is a growing demand of ‘Point-of-care testing (POCT)’ that led to the rapid advancements in technology that will enable the physician to make decision on the bedside. This is making rapid changes in all aspects of health care. One of the major changes in the clinical laboratory has been the implementation and increased use of POCT. This brings the laboratory test to the patient rather than obtaining a specimen from the patient and transporting it to the laboratory for testing. This makes laboratory test results available more rapidly, providing improved patient care. In advanced countries it is applied in numerous situations—nursing homes, physician’s oce, emergency rooms, intensive care units and for bedside testing in hospital wards. The evolution of small, simple-to-use analysers that require only one drop, or less, of specimen has led to widespread POCT implementation. Handheld portable analysers can measure substances such as glucose, haemoglobin, cholesterol and electrolytes. Most require only a drop of blood, usually obtained by nger stick. Thus in the near future many of the routine clinical laboratory tests will be available in remote villages of developing countries although in the urban seing large automated system will continue to function. The clinical laboratories, however, will have to stay involved in making recommendations and compare the results with the classic procedures.
tIme-savIng devIces and KIts
The automated systems are undoubtedly fast, reliable and prove to be cheaper in the long run. However, the initial investment is high and a breakdown can be disastrous. As most of these automated systems are computerized, unfavourable weather, intermient electrical supply and lack of technical repairmen make it hard for the laboratories to decide whether to invest in them or not. Modular systems are more advantageous as spare module can be used in case of breakdown, and the defective machine can be sent for repair. Automated system is protable only when the work load is high.
For smaller laboratories, kits have proved to be protable. Kits must be purchased from reliable companies (Appendix at the end of this volume). The laboratory should purchase such time-saving gadgets as automatic pipeer, diluter, mixer and others to expedite the work. Excessive mechanical work will cause fatigue in the technician and this will result in erroneous results. All technicians must, however, master the manual procedures before they plan for any kind of automation. This reduces the sense of helplessness when the automated system fails.
concLusIon
Automation is a buzzword among clinical laboratories, but it is not a pie in the sky. The future for automation in clinical labs is here. Many clinical laboratory tasks have already been automated, and several manufacturers are oering automated laboratory systems in the marketplace. Automation continues to be the province primarily of hospital laboratories, however, although most large commercial laboratories continue to eschew its usage. Unreliable energy source and instrument breakdown are the main problems of automation in developing countries, other than the high purchase cost and repair. If the laboratory totally
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relies on automation and neglects the wrien manual procedures about what to do, when there’s a system interruption, which inevitably occurs, it will make a big folly. The laboratory must have a backup plan. The patient has no time to wait until the machine gets xed.
Technology enables automation, which in turn, drives ecient laboratories. As automation has become an indispensable part of modern laboratories, its adoption varies by geography. Developing countries tend to take a longer time to adapt the oncoming changes and should select from the large array of automated systems to suit their own conditions. The ideal automated system for the laboratories of developing countries ought to be simple, trouble­free, low-maintenance and reliable. It ought to be able to function with basic chemicals and versatile enough to switch to manual system, if necessary, without sacricing the accuracy of the results. In developing countries, a good backup system must be thought well before the laboratory adopts an automated system. Use of the computers and computer-assisted process control is encouraged and should be leveraged to drive productivity and achieve eciency.
Medical Laboratory Technology: Volume 3
revIew QuestIons
1. What is the dierence between continuous ow analysis and discrete analysis? Name
one instrument in each of these automated systems.
2. Why is it that the centrifugal fast analyser is chosen for enzyme assays?
3. How is the protein interference minimized by the automated systems?
4. Why is it that,the discrete system is preferred over the continuous ow system in the
laboratories of developing countries?
5. What are the diculties you anticipate in computerizing a laboratory located in a devel-
oping country?
6. Dene: CPU, Byte, Hardware, Soware and Algorithm.
7. What are the main components of a computer and what are their functions?
8. If you are asked to install a computer in the laboratory where will you place it?
9. List the most important components of an AutoAnalyzer and state their functions.
10. Why does the AutoAnalyzer introduce air bubbles into the owing stream of the sam-
ple and reagent and how are the air bubbles removed before the detector reads the colour of the solution?
11. What are the functions of the microcomputer system in the Clinical Corona?

Routine Biochemical Test Procedures

Kanai L Mukherjee, Chhotelaal Pande and Rohini Chakravarthy
Chapter Outline
• Introduction
• Routine Diagnostic Tests in Clinical Chemistry
• Blood Glucose
▪ Glucose Assay by o-toluidine Method
▪ Glucose Oxidase (enzymatic) Method
▪ Glycated or Glycosylated Haemoglobin (HbA1c)
• Serum Protein
▪ Total Protein in Serum
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▪ Serum Albumin
▪ Myoglobin
▪ Troponin
• Blood Urea Nitrogen (BUN)
• Uric Acid
• Creatinine
• Bilirubin
▪ Total Bilirubin
▪ Total and Conjugated Bilirubin
• Diagnostic Enzymology
▪ Measurement of Enzyme Activity
▪ Routine Analysis of Diagnostic Enzymes
• Brain Natriuretic Peptide (BNP)
• Lipid Prole
▪ Cholesterol
▪ Triglycerides
▪ Subgroups of Cholesterol
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