Table Of Content

The English Language Book Society publishes low-priced, unabridged editions of important British textbooks. Below is a list of some other medical books available under the ELBS imprint. Anderson Muir's Textbook ofPathology Edward Arnold Baron A Short Textbook of Chemical Pathology Hodder & Stoughton Cheesbrough and McArthur A Laboratory Manual for Rural Tropical Hospitals Churchill Livingstone Freeman and Bracegirdle An Atlas of Histology Heinemann Educational King A Medical Laboratory for Developing Countries Oxford University Press Mason and Swash Hutchison's Clinical Methods Bailliere Tindall Morley Paediatric Priorities in the Developing World Butterworths Ogilvie Chamberlain's Symptoms and Signs in Clinical Medicine John Wright Roitt Essential Immunology Blackwell Scientific Thompson A Short Textbook ofHaematology Pitman Medical Introduction to Medical Laboratory Technology Fifth Edition F. J. B A K E R F . I . M . L . S . , F I S T . R. E. SILVERTON F.I.M.L.S., L.I.Biol. With the collaboration of D. C A N N I N G , f l m l s . (Haematology and Blood Transfusion) D. KILSHAW, F L M L S . (Clinical Chemistry) J . LAW, F L M L S . (Histology) R. SHANNON, M.Phii., f . l m . l . s . (Microbiology) English Language Book Society/Butterworths Butterworth & Co (Publishers) Ltd 88 Kingsway, London WC23 6AB © Butterworth & Co (Publishers) Ltd 1976 All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, including photocopying and recording, without the written permission of the copyright holder, application for which should be addressed to the Publishers. Such written permission must also be obtained before any part of this publication is stored in a retrieval system of any nature. First published 1954 Second edition 1957 Revised 1960 Reprinted 1961 Third edition 1962 Reprinted 1964,1965 Fourth edition 1966 Reprinted 1968, 1969, 1970, 1971, 1972, 1974, January 1975, August 1975 Fifth edition 1976 Reprinted 1978,1980, 1982 ELBS edition first published 1978 Reprinted 1980,1982,1985 ISBN 0 407 00154 9 Cataloging in Publication Data Baker, Francis Joseph. Introduction to medical laboratory technology. Bibliography: p. Includes index I. Medical laboratories—Technique. I. Silverton, R. E., joint author. II. Tide. III. Title: Medical laboratory technology. RB37.B28 1976 616,07'5 ISBN 0-407-00154-9 Printed in Great Britain by Butler & Tanner Ltd, Frome and London Preface During the twenty-two years which have elapsed since this work was first published, laboratory medicine has undergone many changes. These changes relate not only to the more specialized and sophisticated techniques now used, but also to fundamental changes within the profession itself. Automation, data processing and other technical advances have all helped to bring about not only procedural change but changes in outlook. In the United Kingdom, the name of the professional body has been changed from the Institute of Medical Laboratory Technology to the Institute of Medical Laboratory Sciences (IMLS). This in itself reflects the progress which has been made. The examination structure for medical laboratory scientists has similarly been changed, and currently a National Certificate pathway is the common route to Associateship of the I M L S and State Registra tion. Graduates are entering the I M L S and the profession in in creasing numbers, and the formation of a College of Pathologists, subsequently the Royal College of Pathologists, are added signs of the need for an understanding of the increasing complexities of modern medical laboratory sciences. These many changes have been borne in mind in preparing the 5th Edition of this work. The contents have been enlarged, up dated and in many instances have been completely re-written. Despite the changes in the layout, contents of the book, and the profession to which it applies, the original title Introduction to Medical Laboratory Technology by which it is universally known, has been purposely retained. A further change that has taken place in recent years is the introduction, particularly into the National Health Service, of SI Units. System.e International d'Unites (SI Units) were approved internationally in 1960 and are becoming generally accepted in science and medicine in Europe and some other parts of the world. A table of SI units, showing the changes in nomenclature and the abbreviations used, will be found at the end of the book (p. 681). It will be seen that we have used SI units throughout the text. PREFACE with the exception of pounds per square inch (psi). This measure ment has been retained as most autoclave gauges are calibrated this way and it seemed pointless, at this moment in time, to adopt the equivalent SI unit, and thereby produce unnecessary com plications. The more common term ml has also been retained in stead of cm'^ for measurement of volume. Because of the increase in specialization, we have again sought the advice of many colleagues in various disciplines throughout the U K but in particular we would like to record our appreciation of the following, who have acted as collaborators in their various specialities: Mr D. Canning, F I M L S Mr D. Kilshaw, F I M L S Mr J. Law, F I M L S Mr R. Shannon, MPhil, F I M L S . We are hopeful that the 5th Edition of hnroduction to Medical Laboratory Technology will be as valuable to the many types of readers as the earlier editions proved to be. Introduction Medical Laboratory Science is a complex subject embracing a number of different disciplines. The post-war years brought a dramatic increase in the use of the laboratory whose role is to assist in the diagnosis, treatment and control of disease. The increased demands on the laboratory inevitably resulted in the introduction of more specialized and sophisticated procedures in cluding mechanization, automation and data processing. To keep abreast with this modern development, a more academic back ground has become necessary for students entering the profession. Entrants into the profession are now required to undergo formal training at a recognized college of further education or to possess a relevant university degree. These examinations or qualifications coupled with satisfactory experience in a recognized laboratory lead to State Registration by the Council for Professions Supplementary to Medicine. The professional body—the Institute of Medical Laboratory Sciences—is responsible for providing the opportunities to qualify by examinations. At the present moment the National Certificate pathway is the main route to qualification. The I M L S have mem bers on the Joint Committees (who are responsible for the cer tificates in Medical Laboratory Subjects/Sciences) and the I M L S appoint the Assessors for these various certificates. Newcomers to the profession are reminded that it is not a pro fession to be taken up lightly. In order to pass the qualifying examinations, many leisure hours will have to be devoted to study, and, even when qualified, the medical laboratory scientist will still have to keep abreast with modern developments and trends by the regular reading of the appropriate journals. The late arrival of a specimen in the laboratory, the occurrence of which is by no means infrequent, may also necessitate working after hours and sacrific ing private arrangements. Considerable satisfaction, however, will be derived not only from the interesting nature of the work, but also from the knowledge that the duties undertaken during each working day are for the benefit of the community. The importance of his work and his obligations to the patient must ÷ INTRODUCTION therefore be remembered at all times and placed before any per sonal consideration. The Medical Laboratory Technician's Board of the Professions Supplementary to Medicine issued a statement to all registered practitioners of medical laboratory sciences which is reproduced here with their approval. No registered medical laboratory technician should: 1. Hold himself out as a person, who by training and experi ence, is professionally qualified to diagnose or treat disease in man or animal. 2 Knowingly accept, obtain, assist in obtaining or report on any specimen for the purpose of the diagnosis and/or treat ment of disease, or make any investigation for those purposes unless the diagnosis and/or treatment are to be performed by^a registered medical, dental, or veterinary practitioner. 3. Knowingly disclose to any patient or to any other un authorised person the result of any investigations or any other information of a personal or confidential nature gained in the course of practice of his profession. 4. Advertise, whether directly or indirectly, or associate him self in any way with advertisement for the purpose of obtain ing specimens for laboratory investigations. 5. Knowingly falsify or suppress a report of any laboratory investigation with which he may be concerned. 1 G L A S S W A R E Glassware is widely used in medical laboratories, and it is essential to become thoroughly familiar with the common varieties. This chapter is concerned only with some of the more general types, and specialized equipment is not considered. Composition of glass Laboratory glassware is usually manufactured from borosilicate glass, a material developed to conform to certain well-defined characteristics. It is resistant to the action of chemicals with the exception of hydrofluoric and phosphoric acid and is made to with stand mechanical breakage and a sudden change of temperature. Resistance to thermal shock necessitates a low coefficient of thermal expansion. Glassware produced from the soda-lime type of glass does not meet this requirement and is easily broken by the mechanical stress produced by a sudden change of tempera ture. Hardened glass, such as Pyrex, has a low soda content and is manufactured especially to resist thermal shock. The walls of the vessels are generally thicker than those made from soda-lime glass and the low soda content increases the chemical durability of the glass. With the less expensive soda-lime glassware, however, free soda is present on the walls, and must be neutralized before use. The main ingredients of borosilicate glass are as follows: Per cent Silica (SiOp 80.6 Sodiimi oxide (NagO) 4.15 Boric oxide (β ρ^) 12.6 Aluminium oxide (AlgOg) 2.2 General Laboratory Glassware and Apparatus 4 GENERAL LABORATORY GLASSWARE AND APPARATUS CARE OF GLASSWARE All glassware must be handled carefully. Breakages can sometimes be dangerous, and they may result in the loss of valuable and irre placeable material. Certain precautions must be observed. 1. Flasks and beakers should be placed on a gauze mat when they are heated over a bunsen flame. 2. Test-tubes exposed to a naked flame should be made of heat- resistant glass (such as Pyrex). 3. If liquids are to be heated in a bath of boiling water, the glass containers used should be heat-resistant. It is safer to im merse the containers in warm water, which is then brought to the boil, than to plunge them directly into boiling water. Similarly, sudden cooling of hot glass should be avoided, un less it is specifically required. 4. When diluting concentrated acids, thin-walled glassware should be used. The heat evolved by the procedure often cracks thick glass. 5. Heat expansion is liable to crack bottles if their caps are screwed on tightly. I f heat is to be applied, flasks held in retort stands should not be tightly clamped. 6. Containers and their corresponding ground-glass stoppers should be numbered, to ensure correct matching when stop pers are replaced. When these bottles or flasks are being used, the stoppers should be laid on clean filter paper, to avoid scratching them. Cleaning of glassware GENERAL GLASSWARE The cleaning of all glassware is simplified by rinsing in tap water immediately after use. Contaminated material, however, must always be sterilized before cleaning is commenced. New glassware may be cleaned by washing in a detergent such as Pyroneg and then rinsed thoroughly in tap water. Soda-lime glassware should have the free alkali neutralized by standing the glass in a 5 per cent solution of hydrochloric acid. This is followed by several rinses in tap water and in distilled water. If it is desired simply to neutralize free alkali given off" by new glassware, it may be steeped in 1 per cent hydrochloric acid for several hours. This is followed by thorough rinsing. The glass is then dried in a hot- air oven. To test that the free alkali has been neutralized, autoclave the glassware in neutral distilled water, and when cool, check the GENERAL LABORATORY GLASSWARE AND APPARATUS 5 pH of the water. If excess alkali has been given off (the pH is high, seep. 131) re-steep the glassware in the hydrochloric acid. If free alkali still persists after several treatments the glassware should be discarded. Slides required for blood films must be absolutely grease-free. They are soaked overnight in dichromate cleaning fluid (see below) or in nitric acid. After thorough rinsing, they may be stored in methylated spirit until required. For general puφ oses, new slides may be storqd directly in spirit. It is safer not to reclaim slides used for films of tuberculous material. BIOCHEMICAL GLASSWARE Chemical cleaning is necessary for the following reasons. 1. Traces of reagents left in tubes and containers may interfere with later chemical investigations, for example the ö-tolidine test for occult blood may be positive if the glassware contains even minute traces of dried blood. 2. Air bubbles may be trapped between greasy surfaces and contained liquid, resulting in inaccurate volumetric read ings. Procedure for rendering glassware chemically clean 1. Preparation of cleaning fluid: Potassium dichromate 10 g Concentrated sulphuric acid 25 ml Distilled water 75 ml Grind the dichromate crystals in a pestle and mortar, and add the powder to the distilled water in a heat-resistant flask. Pour in the acid very slowly. The heat evolved hastens the dissolving of the potassium dichromate. Note—This fluid should be handled with caution, rubber gloves and apron being worn to protect the hands and clothes and an eyeshield to protect the eyes. If clothes or skin are splashed with the fluid, they should imme diately be washed in water, and any residual acid neutralized with a weak alkali. This, in turn, is washed off with tap water. After repeated use, the colour of the fluid may darken. When this occurs, fresh fluid should be prepared. 2. Steep the glassware in the cleaning mixture for several hours. 3. Remove the glassware, and wash it thoroughly in tap water, to remove all traces of acid and preferably leave in fresh water overnight. 6 GENERAL LABORATORY GLASSWARE AND APPARi^TUS 4. Rinse twice in distilled water. 5. After allowing surplus water to drain off, dry the glassware in a hot-air oven. Procedure for cleaning glassware with detergents The use of detergents for cleaning glassware is becoming increas ingly popular in medical laboratories. These detergents, which are available in either liquid or powder form, owe their cleansing action to the manner in which they reduce the interfacial tension of water with that of oily or greasy substances. Detergents possess the following advantages over ordinary soaps: 1. Their cleansing action is unaffected by the temperature of the water. 2. They are equally efficient in water which is either slightly alkaline or slightly acid. 3. They have no coagulative action on proteins. One serious disadvantage of some detergents is their haemolytic action on red blood cells, the slightest trace of detergent being capable of producing haemolysis. This point must always be borne in mind, particularly with glassware destined to be used for haematology or blood transfusion work. The following procedure should be adopted when using detergents for cleaning glassware: 1. Rinse the glassware thoroughly in cold tap water. 2. Place in the detergent solution and brush thoroughly. 3. Wash thoroughly in running tap water. 4. Rinse three times in distilled water, using fresh distilled water for each rinse. 5. Drain off excess water and dry in the hot-air oven. Glassware dried in the hot-air oven should be packed, mouth down wards, in metal baskets, the bottoms of which are lined with thick blotting paper. Recommended detergents are Decon,* Decon 90 and R B S 2 5 | . These are phosphate-free, surface-active agents suitable for clean ing all glassware and plastics including those contaminated with radioactive material. It has been stated that accurately calibrated volumetric glass ware should never be heated in the oven as the expansion and con- * Obtainable from Decon Laboratories Ltd, Ellen Street, Portslade, Brighton, Sussex. t Obtainable from Chemical Concentrates Ltd, 41 Webb's Road, London SWl 1. GENERAL LABORATORY GLASSWARE AND APPARATUS 7 traction of glass that occurs may render the graduations in accurate. Some workers, however, have shown that this is not the case and that Grade A glassware may be sterilized in a hot-air oven. Cleaning of pipettes 1. Steep the pipettes overnight in cleaning fluid. 2. The following morning, wash them thoroughly in tap water, preferably leave overnight in fresh water, and rinse in dis tilled water. T o facilitate washing, connect the pipette to a water pump, using rubber tubing of suitable bore, and suck tap water through for several seconds. Follow this with two or three rinses of hot distilled water. 3. Dry the pipette with two or three brief rinses of acetone. Drying is best effected by sucking through small volumes of acetone and air successively. Repeat this procedure until the internal surface is quite dry. Alternatively an electrically heated pipette dryer may be used. 4. Wipe the outside of the pipette. 5. T o avoid breakage, store the pipettes in drawers lined with lint. It is convenient to fit the drawers with separate com partments for each size and type of pipette. Note—Immediately after use, pipettes should be rinsed in tap water, especially when they have held proteinous fluid, for example blood. Should the pipette be heavily contaminated with such material, it may be cleaned by standing it in a strong solution of caustic soda. This treatment should not be prolonged, as the alkali dissolves glass and may cause an alteration in contained volume. A pipette which has been used for measuring stain can often be cleaned rapidly by rinsing it through with hydrochloric acid. The cleaning procedures described above do not apply to pasteur pipettes. After use with infected material, these are placed in a disinfectant solution. Standardized glassware Apparatus used for the measurement of liquid volume, for example, pipettes, burettes, volumetric flasks and cylinders, are divided into three grades, depending on the accuracy of calibra tion. The limits of these grades are defined by the British Standards Institution, and the manufacturers mark each piece of standar dized glassware with the appropriate symbol. The maker's assurance, however, is the only guarantee that the product 8 GENERAL LABORATORY GLASSWARE AND APPARATUS Table 1.1 TABLE OF TOLERANCES FOR DELIVERY PIPETTES (BULB TYPE) Capacity in ml 2 10 20 50 Time of outflow seconds, grade A 7-15 18-25 20-35 25-40 Tolerance ± ml grade A 0.01 0.02 0.02 0.04 Time of outflow seconds, grade Β 7-20 15-40 20-50 25-60 Tolerance ± ml grade Β 0.02 0.04 0.05 0.08 The most accurately calibrated glassware available in Britain carries certificates from the National Physical Laboratory. Each such piece of apparatus is etched with the letters NPL. This glass ware is necessary only for the highest standards of accuracy. Example of the markings on an NPL Class A pipette are: NPLA NPL Class A 5 ml 5 ml volume D20°C Delivery pipette: volume cor rect at 20 X 1 0 + 15 10s to deliver: 15s to drain 32867 Certificate number By international agreement, pipettes and vessels 'to deliver' are marked with the letters Έ χ ', while those designed 'to contain' are marked with the letters 'In'. The delivery time is no longer given and indeed it is recommended that the tip of the jet be kept in contact with the inside of the receiving vessel for approximately 3 s after movement of the meniscus has appeared to cease. General glassware BEAKERS These have capacities of from 5 to 5000 ml. They are usually made of heat-resistant glass, and are available in different shapes. The type most commonly used is the squat form, which is cylindrical and has a spout. There is also a tall form, usually without a spout. Conical and flask-shaped types are available, but these are not widely used. Beakers are often supplied in sets or nests of assorted sizes. Some may be graduated, while others may be made of poly styrene. conforms with the BSI criteria. For example, tolerances laid down for bulb-type pipettes are given in Table 1.1. GENERAL LABORATORY GLASSWARE AND APPARATUS 9 BOTTLES These are made in many shapes. Some of the more general types are described. Reagent bottles are supplied in 25-1000 ml capacities. They are cylindrical, have narrow necks, and are fitted with ground-glass or polythene stoppers. Screw-capped bottles are supplied in 5-1000 ml capacities, and may be round or flat (the 5 ml size is often called a 'bijou' bottle). The caps may be made of metal or plastic. These bottles are used for holding specimens, solutions and media. Metal caps should never be used on bottles containing mercuric chloride, as this sub stance will attack the metal. Winchester quart bottles are of2000 ml capacity, and are available in white or brown glass. They may be fitted with glass stoppers, corks or rubber bungs. They are useful for storing stock solutions and reagents, and for specimens, for example, samples of urine collected over 24 h. Drop bottles are of about 50 ml capacity, and are made in white or brown glass, with a narrow neck and a slotted glass stopper. They are designed for delivery of drops of solutions, such as stains. After use the stoppers should be turned, so that the contents are not open to the air. Polythene bottles^ although made of plastic material, should be mentioned here. They are of various sizes and shapes, and some are fitted with a nozzle, for use as 'wash bottles'. BURETTES These are used for measuring variable quantities of liquid, and are made in capacities of 1-100 ml. They are long graduated tubes of uniform bore and are closed at the lower end by means of a glass stopcock, which should be lightly greased for smooth rota tion. Technique of using burettes 1. Before use, half-fill the burette with distilled water, and allow it to be discharged through the tap. Droplets adhering to the glass indicate a greasy surface. I f this is seen, chemical clean ing is necessary. 2. Rinse the burette two or three times with small volumes of the solution to be measured, discharging the washings through the tap. 3. With the tap closed, clamp the burette in a vertical position. 10 GENERAL LABORATORY GLASSWARE AND APPARATUS and pour in the liquid to be measured through a funnel, until the meniscus rises above the zero mark of the burette. Remove the funnel. 4. Open the stopcock tap until the meniscus of the liquid exactly coincides with the zero mark. Make sure that the tap is completely free of air-bubbles, and that the tip has no droplets adhering to it. The burette is now ready for use. 5. When work with the burette is complete, the fluid is drained out. The burette is rinsed through several times with tap water, and then with distilled water. 6. For storage, the burette is clamped in an inverted position. CENTRIFUGE TUBES These are made of hardened glass, nylon or plastic material that can withstand the centrifugal strain. The bottom of the tubes may be round or conical. The latter type is preferable, because the de posit is concentrated into a smaller volume. Some centrifuge tubes are calibrated up to 10 ml. These markings may be useful, pro vided the graduations have been checked for accuracy. DESICCATORS It may be necessary to dehydrate substances, or to keep them in an anhydrous state. This may be effected by storing them in a desiccator over a water-absorbent chemical, such as anhydrous calcium chloride or phosphorous pentoxide. Evacuation of air in creases the rate of dehydration, and some desiccators are therefore made of glass strong enough to withstand a vacuum. Others are not designed for this purpose, and have no tap in the lid. The tap, when present, and the opposed surfaces of jar and lid are of ground-glass. Technique of using vacuum desiccators 1. Place the substance to be stored in an open container, and rest this on the zinc gauze sheet, which forms a platform above the desiccating chemical. Lubricate the tap and the opposing surfaces of jar and lid with Vaseline, petroleum jelly or stopcock grease, to ensure that all junctions are air tight. 2. Slide the cover onto the jar, and rotate it into position. 3. Open the tap, and connect the outlet to a vacuum pump. If this is of the water type, always insert a trap-bottle between the pump and desiccator to prevent any backflow