WHO REGIONAL PUBLICATIONS, EASTERN MEDITERRANEAN SERIES 2 Basics of Quality Assurance for Intermediate and Peripheral Laboratories Ia WORLD HEALTH ORGANIZATION Regional Office for the Eastern MediterraneanWHO Regional Publications, Eastern Mediterranean Series No. 2 Basics of Quality Assurance for Intermediate and Peripheral Laboratories M.M. EL-NAGEH Regional Adviser, Health Laboratory Services World Health Organization Eastern Mediterranean Region Alexandria, Egypt J. VANDEPITTE Department of Microbiology St Ratael Academic Hospital Leuven, Belgium C.C. HEUCK Health Laboratory Technology and Blood Safety World Health Organization Geneva, Switzerland W. APPEL Central Laboratory St Vincentius Hospitals. Karlsruhe, Germany K. ENGBAEK Department of Clinical Microbiology University of Copenhagen Herlev Hospital, Herlev, Denmark W.N. GIBBS Chief, Health Laboratory Technology and Blood Safety World Health Organization Geneva, Switzerland y \ v “i NX SS World Health Organization Regional Office for the Eastern Mediterranean 1992WHO Library Cataloguing in Publication Data EI- Nageh, Mohamed M Basics of Quality Assurance for Intermediate and Peripheral Laboratories. - by Mohamed M. El-Nageh et al, - Alexandria: WHO Regional Office for the Eastern Mediterranean, 1992. - xviii, 208 p. (WHO Regional Publications, Eastern Mediterranean Series: No.2) 1 Laboratories - Standards - handbooks. 2. Quality assurance, health care. ISBN 92-9021 -160-1 (NLM Class.: W 23) ISSN 1020-04 1X The World Health Organization welcomes requests for permission to reproduce or Uranstate its publications, in part or in full. Applications and enquiries should be addressed to. the Manager, Health and Biomedical Information Programme, World Health Organization, Regional Olfice for the Eastern Mediterranean, P.O, Box 1517, Alexandria, Egypt, who will be glad to provide the latest information on any ges made to the text, plans for new editions, and reprints and (raastutions already © World Health Organization 1992 ions of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention All rights reserved ‘The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the purt of the Sceretariat of the World Health Organization concering the legal status of any country, territory, city or area or of ils authorities, or concerning the delimitation of its frontiers or boundaries. ‘The mention of specilie companies or of certain manufacturers! products does not imply that they are cndorsed or recommended by the World Health Organization in preierence to others of a similar nature that are not mentioned Printed by APC in Alexandra, Fgy ptTABLE OF CONTENTS PREFACE ABBREVIATIONS AND ACRONYMS (INTRODUCTION 1. PRE-ANALYTICAL CONTROL BIOLOGICAL SOURCES OF VARIATION Genetic variation Sex-related variation Age-dependent variation Biorhythm Nutrition Posture of the patient during blood sampling Physical exercise Non-periodic changes SOURCES OF VARIATION IN SPECIMEN COLLECTION, TRANSPORT AND STORAGE BLOOD SPECIMENS Collection of blood specimens Venous blood sampling Capillary blood sampling Preservation of blood specimens Preparation of blood films Fresh wet film Thin blood film - Thick blood film ‘Transport and storage of blood Haemolysis Artificial causes of haemolysis Chemical tests affected by haemolysis Collection and transport of blood for microbial investigations Recommended agents for skin antisepsis prior to blood sampling Venipuncture Selection of the venipuncture site Preparation of the venipuncture site and containers Blood cultures Number of blood cultures Procedure for blood culturing Selection of blood culture media v Aan oePLEURAL FLUID 21 PERITONEAL FLUID 2 CEREBROSPINAL FLUID 23 Interpretation of microscopic and cli | investigation 24 THROAT SWABS 25 Interpretation 2 SPUTUM 27 URINE 2 Urine collection ww Patients not requiring assistance 29 Bedridden patients requiring assistance 30 Intants 30 ‘Transport of urine ai SEXUALLY TRANSMITTED DISEASE SPECIMENS 3 Gonorrhoea 31 Gonococcal urethritis in male BSE Gonococcal cervicitis in females 32 Vaginal discharge 32 Genital ulcers 2 Blood for serology 22 STOOLS 33 2. CONTROL OF LABORATORY INVESTIGATIONS a5 GENERAL MEASURES 36 Calibration 36 Duplicate tests on a patient's specimen 36 Check tests 36. Replicate tests 36 Control chart a7 Patient data 38 Intralaboratory comparison and interlaboratory comparison 38. Control of precision 39 Control of accuracy 39 Control of dry chemistry tests 4 LABORATORY MANUAL 40 SPECIFIC MEASURES 43 CLINICAL CHEMISTRY 43 Blood serum and plasma tests 4 Albumin 43 Alkaline phosphatase 44 Alpha-amylase 45, Aspartate aminotransferase (AST) 46 Bilirubin 47 ViCalcium 48 Glucose 49 Urea 50 Creatinine 50 Protein 2 Plasma iron 53 Urine tests 33 Dry chemistry tests 58 HAEMATOLOGY 60 Haemoglobin 60 Packed cell volume 63 Red blood cell count 65 Total white blood cell count (WBC) 69 Platelet counting u Reticulocyte counting nR Differential blood count 73 Principles for staining techniques 73 Erythrocyte sedimentation rate 7 Investigation of haemoglobinopathies 8 Screening for glucose-6-phosphate dehydrogenase 79 Investigations of haemostatic disorders 80 Bleeding time 80 Prothrombin time 82 Activated partial thromboplastin time sz ‘Thrombin clotting time 83 Fibrinogen 83 Immunohaematology tests 85 CLINICAL MICROBIOLOGY 86 Collection and transport of specimens. 86, Quality control of culture media and reagents 87 Media and reagents prepared by the user 87 Sterility testing 8S Performance testing SN Antimicrobial susceptibility tests ow Antisera and antigens for serology 92 Maintenance and use of stock cultures Ox Long-term preservation 03 Short-term presery alien o8 Reporting of results us SEROLOGY oF MEDICAL PARASITOLOGY 10, Proficiency of personnel Hie vilEquipment 100 Quality control of specimens 101 Macroscopic evaluation 101 Microscopic procedures 101 Faeces 101 Blood 102 Quality controt of stains and reagents 103 Quality control of reports 104 3. DATA HANDLING AND DATA PROCESSING 105, Personal data of patient 105, Record keeping . 108 Outlier test i Delta check 1 Use of computers for control of laboratory performance M3 4. LABORATORY MANAGEMENT AND ORGANIZATION 114 5. USE AND MAINTENANCE OF LABORATORY EQUIPMENT 117 Basic laboratory equipment 17 Flame photometer ANB. Operation and maintenance 120 Photometer and colorimeters Light source Monochromators Filters Cuvettes Detector and multiplier Photometric measurements Calibration Cell counter 128 Microscope 132 Cleaning optics 132 Instrument cleaning 132 Additional precautions to be taken in hot climates 132 Centrifuges 133 VILRefrigerators Maintenance of refrigerators Daily checks Monthly checks Hot oven, autoclave, incubator and water bath Hot oven Autoclave Incubator Waterbath Water purification systems Demineralization Distillation Filtration Pipettes Alternative sample pipettes Autopipettes and dispensers Pipetting Calibration of mechanical micropipettes and dispensers Test tubes pH meters Unpaking a new electrode Calibration of pH meters Measuring the pH of the test solution Balances Maintenance of balauces 6. SAFETY PRECAUTIONS IN THE LABORATORY MANAGEMENT OF THE LABORATORY SAFETY PROGRAMME Safety instructions Reporting of accidents Educations PERSONAL HYGIENE, LABORATORY ENVIRONMENT, PRACTICE AND PROCEDURES SHIPMENT OF SPECIMENS DISPOSAL AND DI CTION OF CONTAMINATED LABORATORY MATERIAL Disposal Disinfection RGENCY PROCEDURI FOR ACCIDENTS 1X 136, 138, 138 138 139 139 140 14d 142 142 142 rey 146 146 147 148 148 Mo 160 160 160 164Abrasions and cuts Fire and thermal burns Chemical hazards Electric hazards Ocular injuries 7. EXTERNAL QUALITY ASSESSMENT ANNEXES: Annex 1. CONVERSION FACTORS FOR COMMON ANALYTES FROM CONVENTIONAL UNITS INTO SI UNITS Annex 2. USEFUL SOLUTIONS AND STAINS IN HAEMATOLOGY Annex 3, DESCRIPTIVE TERMS OF THE MORPHOLOGY OF RED BLOOD CELLS Annex 4, STATISTICS OF QUALITY CONTROL GLOSSARY REFERENCES AND RECOMMENDED READINGS 164 165 165 166 167 169 Amt 173 175 181 191 198PREFACE During the last few years peripheral extension of health laboratory services. located at front-line health care delivery facitities with increased population coverage. has been achieved in several countries of the Fastern Mediterranean Region (EMR). Obviously. despite such extension and improvements, there is still a number of problems 1 be solved. One of these is that the quality of laboratory performance is often not of the standard required, Laboratory services will improve the quality of health care only if quality assurance is accomplished, by solving problems that limit the improvement of laboratory performan evident that high-quality performance of laboratory servives will considerably contibute © effectiveness in both financial and heath re terms Realizing the importance of quality assurance in laboratory performance. WHO's Eastern Mediterranean Regional Ollie (EMRO) has helped to back up national efforts to establish quality assurance programmes in the Region, EMRO suggested to every country of the Region that a national centre of reference, responsible for the implementation and supervision of an external quality assessment scheme, be nominated. Each centre should be headed by a motivated, qualified and well-trained prolessional who should also be ae health laboratories participating in the programme. WHO collaborating centres are prepared to arrange for individual short periods of trainin table to most of the on running a successful programme. This publication is intended for laboratory specialists. technologists. and_ technicians conducting internal quality control who can contribute to the improvement of health laboratories’ performance by applying the principles of quality assurance. The authors are grateful for the valuable continuous support received from Dr Hussein A. Gevairy, Regional Director for the Eastern Mediterranean Region, and Dy, M. ff. Khayat Director, Programme Management, for his continous encouragemeat and support, Our thanks are also due to Dr André Deom (Switzerland), Dr René Dybkaer (Denmark). Dr Anders Kallner (Sweden), Dr A. M. Lewis (United Kingdom) and Dr P. da Fonesca-Wollheim (Germany), for reviewing the draft and for constructive suggestions.ABBREVIATIONS AND ACRONYMS BCR BIPM (CIPM) CE CEC CEN CENELEC cIPM ECCLS EEC EFTA, EMR EMRO EOTC FICC ICSH IEC IFCC ISBT Iso ISTH TUPAC NCCLS, OECD. Bureau Communautaire de Référence (Bruxelles) Bureau International des Poids et Mesures (Sevre/P: Intemational Bureau of Weights and Measures Couneil of Europe Conseil de l'Europe (Bruxelles) Commission of the European Community Commission Européenne Communautaire (Bruxelles) Comité Européen de Normalisation (Bruxelles) European Committee for Standardisation Comité Européen de Normalisation Electrotechnique (Bruxclles) European Committee for Standardization in Blectrotechnies See BIPM. European Committee (Council) for Clinical Laboratory Standards European Economic Communities (Common Market) Le Marché Commun Européen European Free Trade Association (Geneva) Eastern Mediterranean Region Eastem Mediterranean Regional Office a for Testing and Certific: n (Brussels) international Committee for Standardization in Haematology Intemational Electrotechnical Commission (Geneva) Commission Intemationale Eiectrotechnique International Federation of Clinical Chemistry International Society of Blood Transfusion International Organisation for Standardization (Geneva) International Society for Thrombosis and Haemostasis International Union of Pure and Applied Chemistry (Baste) National Committee for Clinical Laboratory Standards Organization for Feonomic Cooperation and Development (Paris) XiOIML OMS REMCO UICPA WHO ACD AHG APTT ALT ASLO AST BCG *c CK em cone CRP CSF CTA DIC DIN dL EDTA EQA ESR °F FDPs. fL G6PD Organisation Internationale de Métrologie Légale (Sevres/Paris) International Organisation of Legal Metrology Organisation Mondiale de $a Santé Reference Materials Council Commitiee, ISO (Geneva) See HUPAC World Health Organization (Geneva) Scientific abbreviations Acid-citrate dextrose Antihuman globulin Activated partial thromboplastin tinte Alanine aminotransferase Antistreptalysine O Aspartate aminotransferase Bromoeresol green Celsius degree Creatinine phosphokinase Centimetre Concentration C-reactive protein Cerebrospinal fluid Cystine trypticase agar Day Disseminated intravaseular coagulation Deutsche Industrie Norm Decilitre Ethylene diamine tetra-acetate External quality assessment Erythrocyte sedimentation rate Fahrenheit degree Fibrin(ogen) degradation products Femtolitre Gtucose-6-phosphate dehydrogenase Gram XIVIgG Ig IgM Iac INR Gravitation force Weight/volume (w/v) Hour Haemoglobin Oxygenated haemoglobin, oxyhaemoglobin Haematocrit Haemoglobincyanide Human immunodeficiency virus Immunoglobulin A Immunoglobulin E Immunoglobulin G immunoglobulin D Immunoglobulin M Internal quality control International normalized ratio Kilogram Litre Volume/volume (v/v) Lactate dehydrogenase Decadic logarithm Mean corpuscular haemoglobin Mean corpuscular hacmoglobin concentration Mean corpuscular volume Milligram ion Microgram Millilitre Microlitre. Millimetre Minuic Micrometre Millimoleflitre Micromole/litre licroSiemens Millivolt Nicotinamide adenine dinucleotide Reduced form of NAD Nicotinamide adenine dinucleotide phosphate XVONPG PVA QA a RBC RCF Rh SD SOPS STD ‘TCBS TCT TSA uv VDRL, wBCc VIM IsO/DIS EN prEN O-nitrophenyl-beta-galactopyranoside Packed cell volume Picogram Pro-hydrogenium (negative log of hydrogen ion concentration) Afternoon Prothrombin time Partial thomboplastin Polyvinyl aleohol me Quality assurance Quality control Red biood cell Relative centrifugal force Rhesus factor Standard deviation Standard operating procedures, Sexually transmitted diseases Thiosulfate citrate bile-salt sucrose Thrombin clotting time Tryptic soy agar Unit Uttraviolet Venereal disease research laboratory (test for syphilis) White blood cell Year Standards Vocabulaire international des termes fondamentaux et métrologie International Vocabulary of Basic and General Terms in Metrology raux de Draft Imerational Standard ISO European Standard Norme européenne Proposed European Standard Norme curopéenne de standardisation proposée XVIINTRODUCTION Quality assurance aims at ensuring, for poth laboratory staff and clinicians, that the data provided are relevant and reliable, thus improving trust in laboratory results. As, in practice, it is not possible to assure absolutely that every result will meet the required quality, quality assurance in fact means quality enhancement. Quality assurance involves all measures that can be taken to improve laboratory efficiency and effectiveness, with a view (0 the maximum benefit to the individual and the community, as well as to ensure Laboratory performance with minimal risk for kiboratory workers. Quality assurance should include, besides intemal quality control (IQC) and external quafity assessment (EQA), all elements of its extended concept; these include clinical usefulness of selected tests, patient preparation, sampling, specimen handling, preservation, storage, transport, identification and data processing (including reporting of results, recording and charting, interpretation of results and feedback). This necessitates adopting a broad concept of quality management to identify sources of intralaboratory excellence and 10 include étaining of personnel, supervision (an extremely important clement). supply of information to users (clinicians) and performers (laboratory staff), performance standards, use of reliable reagents, selection and maintenance of appropriate equipment, documentation and biosafety. One very important aspect is. the availability of appropriate documentation: at least one major practical textbook in eaets laboratory medicine speciality not more than five years old, should be available in each laboratory. Professional and technical laboratory personnel should be encouraged to attend meetings, seminars and refresher courses to enable them to update their knowledge and skills. Clinicians repeatedly raise questions about the reliability of the laboratory reports without, sometimes. being aware that reliability of results depends on many para: as well as analytical procedures. Traditionally. concern has been expressed regarding various intralaboratory technical aspects that may afleet the analytical process. The analytical cycle, however, is more complex and includes pre-analytical and post-analytical elements. which may be sources of error that are beyond the control of laboratory staff. This important fact should be borne in mind by laboratory users and performers. Both laboratory staff and clinicians should realise that quality assurance, being their mutual responsibility. cannot be achieved through improving and controlling the analytical process without parallel improvement and control of pre-analytical and pos nalytical factor lytical procedures. For the analytical part of a quality assurance programme, it should be emphasized that the application of good laboratory practice is an essential element, without whi can be achieved. No impact of external quality assessment can be expected without the establishment of an efficient intemal quality control programme (or system). This must be part of routine laboratory work. For this reason, more emphasis is placed in this Manual on interna} quality control. no improvement At bench level. internal quality control is particufarly important: it must include tests using control material and analysis of patients’ data, The main objective is to ensure precision, if possible in agreement with an indicator of truth, Control procedures are used for qualitative as well as qui wtitative procedures, XVIDAnalytical quality control ensures continuity of the following: © equipment reliability @ reagent stability, integrity and efficiency © adequate calibration © procedure reliability in terms of analytical specificity, precision and accuracy Non-analytical quality assurance includes: request specifications © patient preparation @ details of patient and specimen identification © transport and delivery of specimens, The effect of interactions and/or interferences, including drugs, on laboratory results should be remembered; however, detailed information on this subject is beyond the scope of this Manual. XVII1. PRE-ANALYTICAL CONTROL The analyses performed by health laboratories are subject to biologival and technical influences, professional skills and environmental effects. The physiological changes that occur in healthy and diseased subjects are frequent sources of misinterpretation of laboratory results, even when the laboratory investigation has been satisfactorily performed. Not surprisingly, they may give rise to unnecessary discussions between the laboratory and health care personnel and may also lead to further unnecessary investigations. Sometimes even the medical doctor is not aware of the pre-analytical influences. It is therefore very important that collection of specimens be done under standardized conditions. which inelude the preparation of a patient prior to sampling, recording of personal data, c.g. age, sex, and clinical diagnosis and treatment. The laboratory should make a list with detailed instructions for specimen collection to minimize pre-analytical factors that could cause misinterpretation or misunderstanding of laberatory results. Biological sources of variation Achievement of the most accurate measurement of an analyte (substrate concentration, enzyme or hormone activity, panicle counting and coagulation time measurements) is a question of methodology. The result obtained should be nearest to a “true value”. The true value can be measured by sophisticated, so-called " definitive” techniques for simple analytes. but which can rarely be determined by techniques used for routine investigations. Furthermore, the true value has not yet been identified lor a number of the more plex analytes. From a cli al point of view this scientific approach towards laboratory investigations is not always practical. The clinician is interested in being provided with information in a way that allows hinyher to compare the pathophysiologieal situation of a patient with the physiological situation of the healthy state, Such a comparison implies the definition of a healthy state in terms of so-called "reference values" for each analyte. The ranges of these reference valucs, as well as the results obtained from measurements in a patient's specimen are prone to a number of pre-analytical influences, as follows: genetic variation age-related variation biorhythin (short- or long-term) nutrition, posture of the patient during specimen sampling physical activity non-periodic changes smoking,Genetic Variation A number of genetically determined diseases are endemic in certain populations. In these populations the prevalence of the disease may be high, while it is very low in other populations. Some examples are: sickle cell anaemia in African populations © thalassaemia in Mediterranean and Asian populations © lactose intolerance in Asians and Africans. Other genetic disorders are more or less equally distributed in different populations. Examples are: haemophilias A & B familial hypercholesterolaemia phenylketonuria cystinuria Gaucher's disease cystic pancreatic fibrosis (mucovisc hypothyroidism. These diseases develop in consequence of a genetic aberrance which causes an imbalance in the regulation of the biological system of the human body. Clinical symptoms are most evident in homozygotes, who may exhibit evidence of the disease at bisth or in carly childhood. The investigator must therefore be aware of such defects when examining patients, particularly sick children who have entered a clinic for more trivial reasons. In hetcrozygotes clinical signs are less pronounced. They are sometimes not even evident clinically and are often more readily detected by laboratory investigations. Fortunately, a genetic variation does not necessarily result in the development of a disease but may be demonstrable in the different distribution range of values determined from healthy persons in different populations. Examples are: blood group O ¢ haemoglobin variants @ — bisalbuminacmia. More than 300 variants of the haemoglobin molecule have been identified, only a few of which cause clinical symptoms. The exact diagnosis of a genetic defect by laboratory investigations is in some cases a prerequisite for appropriate drug treatment, For example, the antimalarial drug primaquine and sulfonamides will cause haemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Therefore G6PD deficiency should be excluded in a patient prior to treatment with such drugs. Ethnic differences do not have any influence on results obtained from routine laboratory measurements. However, interpretation of results (e.g., white blood cell count and haemoglobin concentration) could be affected by the ethnic origin of the patient.Sex-related Variation ‘The normal distribution range in the concentration of some analytes which are measured in blood and urine is also predetermined by sex. This is most evident for the sex hormones but such differences also exist for more common analytes: the differences of the reference ranges for some of these analytes as observed in an adult Caucasian population are given in Table 1. TABLE 1: Sex-dependent variation of limits of reference values in a Caucasian population ANALYTE MALE FEMALE Erythrocyte sedimentation rate (mm/h) 1-13 1-20 Haemoglobin (mol/L) 81-112 74-9.9 (g/l) (130-180) (120-160) Haematocrit (L/L) 0.40-0.54 0.39-0.51 Uric acid (umol/L) 210-420 150-350 (mg/dL) (3.5-7.1) (2.5-5.9) Alanine aminotransferase”? (U/L) 10-50 10-35 Aspartate amino-transferase'”' {UiL) 10-50 10-35 Gamma-glutamyl-transterase"” (U/L) 5-80 5-50 “Measured at 87°C For the following analytes the sex-dependent differences in concentration are negligible: urea, glucose, alkaline phosphatase, immunoglobulins. Age-dependent Variation Age-dependent changes of concentration of activities occur in a number of haematological and chemical analytes. Most impressive are the differences of results from hlood of a neonate when compared with the normal ranges for adults, such as in the case of bilirubin, glucose, total protein, serum iron, immunoglobulins: IgA, IgE and IgM and erythrocyte sedinentation rate. During adolescence some analytes reach values which could be misinterpreted as highly pathological if the age of the patient is not taken into account, This applies particularly to lymphocytes in childhood, serum atkaline phosphatase activities and CSF protein during adolescence (Table 2). Age-dependent changes in concentration also occur in adults, pointing to the need for a cautious interpretation of laboratory results. Most prominent are changes in scrum creatinine, serum cholesterol and serum phosphate (Fig. 1).TABLE 2: Age-dependent limits of reference values for a Caucasian population ANALYTE NEONATE — 1-14 YEARS ADULTS: WBC (x 109/L) 9-30 5-145 4.3-10.8 Lymphocytes (%) 30-35 40-48 25-35 Haemaglobin (g/L) 145-245 120-180 HbAy 20% 97% HbF 80% 1% Cat+ (umol/L) 1.75-2.70 2.10-2.70 2.1-2.70 K+ (mmol/L) 5.0-10.0(1) 3.2-5.4 3.6-5.0 Creatinine ( umol/L) [Jatté reaction] 35-60 50-120 Aspartate aminotransferase (U/L)'2) 10-50 - 10-50 Alanine aminotransferase (U/L)? <25 - 10-50 Gamma-glutamyitransferase (U/L)'?) <210 <20 <50 Alkaline phosphatase (U/L)'?-) <420 <960 <275 “Umbitical ve 2 are hou (without AMP butler) 200 serum creatinine ee eo serum cholesterol s 100 me serum phosphate — \ alkaline phosphatase 0 10 8620 6300640 5060 70 age Grs) FIG.1: Age-dependent changes of mean values in some serum analytesBiorhythm Even in an individual the concentrations of certain analytes may change following a biorhythm. Typical examples following a circadian (= daily) rhythm are hormones. e.g. corticosteroid hormones and thyroid hormones. Discussion of these is beyond the scope of this Manual. For serum iron, concentration is highest at noon and lowest in the evening. Although such circadian undulations occur they are less important for the interpretation of a laboratory result than for the monitoring of the clinical situation of a patient. This is particularly true for serum iron, the concentration of which is also influenced by a number of other factors such as sand infection sure: Evidence of biorhythmie changes with longer periodicity is demonstrable in females. During the menstrual eycle serum cholesterol concentration is lowest at the time of ovulation, Plasma creatinine and urea concentrations increase during the phase of menstruation, while the concentration of serum iron, serum phosphate and serum protein is lower. These changes are known as lunar-monthly rhythms. Climatic and meteorological conditions and geographical location (altitude from mean sea level) show no effect on reference ranges, except for haematocrit and erythrocyte count: residents at high altitudes have higher erythrocyte counts and haemoglobin levels than those at sea level. Nutrition ‘The nutritional status of the patient may sometimes strongly affect the concentration of a number of analytes in blood. it may even be pertinent to keep him/her on a special diet prior to the investigation of blood, so as to ohtain more accurate information about metabolic status. Although such preparation of a patient is usually neglected in primary health care, nutritional status must be recorded to allow for an accurate interpretation of results for certain analytes, otherwise a healthy situation may be falsely taken as pathological, Table 3 lists some analytes which are particularly affected by the nutritional state. Ingestion of large volumes of water may result in falsely normal urine giucose concentration: on the other hand borderline dehydration may cause apparently elevated urine glucose concentration, In some subjects ethanol ingestion acutely changes activities of alanine aminotranslerase (ALT) and aspartate aminotransferase (AST). Smoking does not usually influence results of common analytes: however, Hb is increased in chronic smokers. Elevated body weight influences blood and plasma concentrations of common analytes only to a minor degree; it affects, however, results of measurement of blood plasma volume. Posture of the patient during blood sampling A source of change in concentrations of analytes in blood is the position of the patient during blood sampling. Changes in concentration or activity are more pronounced when bloodTABLE 3: Changes in concentration or activity of analytes from a patient in a different nutritional state ANALYTE Serum triglyceride Serum phosphate Serum urea Serum glucose Serum calcium Serum iron Gamma-glutamy|-transferase Aspartate aminotransterase (AST) Urine ketone bodies Urine osmolality Urine pH CHAN elevated after fat-rich meal elevated after ingestion of large quantities of milk elevated after meat ingestion elevated after carbohydrate ingestion elevated after ingestion of large quantities of milk lower after continuous ingestion of iron-deficient diet elevated after alcohol ingestion elevated after alcohol ingestion increased during fasting decreased after ingestion of large volumes of ‘water elevated after ingestion of vegetables TABLE 4: Changes of concentration or activity in blood obtained from a patient who had changed from a horizontal to an upright position ANALYTE CHANGE Urea - 3% Kr + 3% Cat + 4% Creatinine + 5% Protein + 10% Serum iron + 7% AST + 18% ALT + 15% Alkaline phosphatase +12% Cholesterol + 8% Haematocrit + 10% Albumin + 10%is taken from a healthy person changing position from horizontal to upright, than changes resulting from technica) factors during investigation in a laboratory with good practice (Table 4), A change from the upright to the horizontal position may result in a change in concentration or activity of certain analytes up to 156. Cell concentrations are also affected by the posture of the patient: the Hb concentration and PCV are lower in the supine than in the erect patient. Therefore, serial blood samples should be collected from the patient keeping the same position on each occasion. Although such influences are usually not important for the diagnosis of a disease, they need to be considered for repetitive investigations of a patient within a short period of time. Such changes may be even greater in a diseased patient Physical exercise Physical exercise may cause pronounced changes in the activity of enzymes occurring in muscle. Creatine phosphokinase activity (CK) as well as aspartate transaminase increase markedly after physical exercise, and therefore may mimic the picture of myocardial infarction in terms of laboratory results. Haemocyturia and haemoglobinuria may develop after heavy exercise in long-distance runners, after horse-riding and all kinds of hard physical exercise. The activities of some coagulation factors also increase following exercise. Prolonged physical exercise has been found to considerably decrease the levels of various hormones. including epinephrine and sexual hormones. Increases of Hb concentration and PCV and of the RBC, WBC and platelets in dehydrated patients are due to the decrease in plasma volume. There is an increase in the number of circulating neutrophils during and following physical exercise which may cause a neutrophilic leukocytosis. They may mask anaemia, leucopenia or thrombocytopenia, or may result in spurious polyeythaemia, leukocytosis or thrombocytosis. Conversely, they are decreased when plasma volume is increased. Non-periodic changes ‘The occurrence of a non-periodie change, such as pregnancy. may alter the reference values for a number of common analytes, such as transaminase activities, serum lipids and hormones. In the lust trimester of pregnancy alkaline phosphatase activities are above the range prevailing in a non-pregnant state. There is usually an inerease in the number of neutrophils during pregnancy — sometimes, there is even a neutrophilic leukocytosis. The ESR also increases during pregnancy. On the other hand, the Hb concentration, PCY and RBC decrease. due at least partially to an increase in plasma volume. The activities of some of the coagulation factors increase during pregnancy The effect of interactions and/or interferences. especially by drugs, on faboratory results has to be remembered.Sources of variation in specimen collection, transport and storage The most frequent source affecting laboratory analysis in a well-functioning laboratory is not the laboratory investigation itself but specimen preparation and errors in identification or labelling. Changes in the composition of a specimen can be caused dui collection transportation centrifugation storage Because of alterations in the specimen prior to measurement, the clinical state of the paticnt will not necessarily be reflected by the results of laboratory investigations. despite correct laboratory performance, The following section describes propet ways of collection and transportation of the specimens fisted below and possible alterations Blood specimens for chemical, microscopical and microbiological investigation Pleural fluid Peritoneal fluid Cerebrospinal fluid Throat swabs Sputum Urine Specimens from patients with sexually transmitted diseases Stools Blood specimens The vascular system consists of three parts, all of which may be used for blood collection: the venous system the arterial system the capillary system While the concentration of some analytes does not change from one body compartment to another, for others it may change as a consequence of a change in metabolism or of the distribution between the body compartments. For example, the concentrations of blood gases in arterial blood differ from thase in venous blood, Oxygen concentrations are higher in arterial blood, while carbon dioxide concentrations are higher in venous blood. Arterial glucose concentrations are higher than venous glucose concentrations. Protein concentrations. in capillary blood are higher than in venous blood.Collection of blood specimens ‘The simplest technique for blood collection, capillary puncture, is strongly affected by technical sources of error, Furthermore, only small volumes of blood can be obtained. which on the other hand may be advantageous, Despite the more pronounced variance of results. ary blood sampling in newborns, and in adults whose venous system is difficult to aeeess, has been found satisfactory from a clinical point of view, provided that appropriate salety precautions in capillary puncture are maintained. Venous blood samples are preferable for determination of platelet counts be > to the puncture wound made for collecting capillary samples. Arterial blood sampling may cause more complications and is, slightly more difficult 10 perform than venous blood sampling. whieh is the technique of choice for adults. Arterial blood sampling may only be done by a medival doctor, Therefore it will not bed in this Manual use platelets adhe: be dese Venous blood sampling Supplies Needles, bore size 19, 20, 21 (the last is preferred for coagulation studies) mL. 10 mL, jauze pad Tourniquet Alcohol (isopropanol) 70% eccee Technique {. Prior to blood sampling it should be asc for 12 hours. 2. The patient should sit on a chair so that he/she can comfortably stretch horizontally the left or right arm. This applies also to a patient in horizontal position. The elbow mxey be Supported by an armrest Ascertain whether the patient is right or left handed as preferably the working arm (most often the right arm) should be selected Jar blood collection, since on this side veins are easily accessable, Tie the gourniquet around the arm about 10 cm above the elbow. The collection site is most often the median elbow vein, but other veins may also be considered depending on their detectability. in their filled state, in the angle of the elbow. 4. Palpate the filled vein with your clean finger and cleanse the site for vein puncture with a sterile gauze pad soaked with 70% alcohol.* The tourniquet should not be left on the arm [or more than 2 minutes, because haemostasis for loo long will disrupt the normal balance of the chemical and cellular elements in blood to be drawn. Attach a sterile needle firmly to a sterile syringe and puncture the needle at a low angle {about 15-25") to the skin surface. the tip of the needle directed towards the tourniquet. The puncture should be dane with a quick movement in the direction of jtined whether or not the paticnt has fasted * Special precautions for blood sampling for bacterial culture are described below,10 the anatomical course of the vein to avoid injury to the interior wall of the vessel, which would cause pain to the patient The person collecting the blood sample must ensure that clotting does not eccur du collection (e.g. duc 1 lengthy procedure and/or to multiple attempts with the same needle), and that there is adequate blood flow without turbulence. Syringes and needles must be clean and dry, although blood for coagulation studies is. often collected in a syringe containing anticoagulant 6. Fill the syringe by pulling the plunger slowly to maintain a continuous blood flow, but avoid development of strong negative pressure in the syringe cha cause collapse of the vein, foaming and haemolysis of red blood cells 7. After the syringe is filled, place a sterile dry gauze pad under the needle still sticking in the vein. Open the tourniquet to allow blood (o flow freely, Do not pulll the syringe and needle out prior to opening the tourniquet. 8. Withdraw the syringe with the needle from the vein with a quick movement: immediately press the gauze pad directly on to the puncture site and maintain pressure (e.g. a finger on the gauze pad) for a while. 9. Remove the needle from the syringe. 10. Expel the blood slowly out of the syringe into the prepared collection tube containing the anticoagulant by pushing the plunger. Turn the tube gently to mix the blood with the anticlotting reagent when used (EDTA for cell counting or sodium citrate for coagulation analysis). If glass syringes iste used, the required amount of citrate solution has to be drawn into the syringe before aspirating the blood. Rapid expulsion of the blood may cause haemolysis, 11. Cover the tube with a stopper for further transport 12, Label the tube with identification data of the patient, date and time of sampling and special remarks (if necessary) such ay "urgent request” of "infectious specimen”. Sometimes it is preferable to label the tubes before beginning blood collection. nber. which may PREVENTING HAEMATOMA DURING VENIPUNCTURE Use preferably veins in the elbow area, and only major veins Be careful that the bevel of the needle is fully inside the vein Be careful not to transverse the vein Loosen the tourniquet and ensure haemostasis with a dry sterile cotton ball before pulling out the needle . Ideally, a two-syringe technique should be-used for blood collected for coagulation studies: the first 2-3 mL of blood (or more depending on the number and type of other investigations required) are collected in the first syringe, and the blood for coagulation studies-is collected in the second syringe which contains the appropriate volume of sodium citrate. The second syringe should be silivonized or plastic. Blood samples for investigations should preferably not be taken from intravenous lines but, if this is unavoidable, care must be taken to ensure that the sample is not diluted by thean intravenous fluids being administered, It is particularly important to ensure that blood samples obtained for coagulation studies do not contain heparin from intravenous lines. Sometimes blood is collected directly into a vacuum tube. If the blood has been collected in a syringe, the needle is removed before the tube is filled. The syringe is placed below the rim of the tube and the blood is expelled gently down the side. If the sample iy w be anticoagulated, the blood is mixed gently with anticoagulant by repeated inversion, These manoeuvres reduce the risk of frothing. of haemolysis and of activation of clotting factors, Capillary blood sampling Capillary blood sampling can be done when only small volumes of blood need to be collected, {t is a particularly useful method for blood collection in neonates and babies. but it can also be used for adolescents and adults. Capillary blood samples are sometimes used for determination of Hb concentration, PCV, WBC, RBC. platelet count or reticulocyte count, Errors due to dilution by interstitial fluid are avoided by ensuring free Now of blood without squeezing and by removing the first drop of blood before collecting the sample Sites for capillary blood collection Ear-tips in adults and children Fingertips in adults and children Fingertips in neonates Big toe and medial and lateral positions of the plantar surface of the foot of a neonate (Fig, 2), FIG.2: Sites for capillary blood collection12 Supplies Cotton batts © Capillary tubes «Ethanol or isopropanol, 70% © Lancet or needle, sterile Sterile gauze pad Technique 1, Warin the site of puncture or apply a special cream which increases blood flow after a few minutes of action. The increase in blood flow allows accumulation of capillary blood which has a composition similar to that of arterial blood at the site of puncture. Hyperaemia will also develop after rubbing the skin or after contact with warm water or warm dry gauze, . Cleanse the puncture site with a sterile gauze pad soaked with 70% alcohol. 3. Dry the puncture site with a sterile gauze pad, so that residual alcohol will not mix with outflowing blood: otherwise haemolysis may occur. 4, Puncture the site with a sterile lancet and collect the outflowing blood in a capillary tube, the size of which will depend on the volume of blood required for the planned laboratory investigation (usually between 20 and 100 mL). 5. Alter blood collection . if necessary, cover the punctured site with a dry sterile gauze pad to stop further bleeding Remember To avoid infection do not puncture the same site twice Preservation of blood specimen: The preferred anticoagulant for most haematological investigations (e.g. of Hb concentration, PCV, WBC, RBC, reticulocyte count or platelet count) is a dry EDTA (ethylene diamine tetra-acetate) salt, preferably di-potassium EDTA. (The term "EDTA" is used from now on to refer to all EDTA salts.) The final concentration of anticoagulant should be 1.5 (4.0.3) mg/mL. blood. If insufficient volume of blood has been added to the tube the final concentration of anticoagulant will be increased. This may cause an erroneously low PVC, particularly if the final concentration is more than 2 mg/mL. blood, {t may also cause cellular artefacts due to shrinkage of blood cells in films made from the blood samples. Sodium citrate (0.11 mol/L) is the anticoagulant used most widely for collection of blood for coagulation studies, The ratio of blood to anticoagulant is 9:1, For erythrocyte sedimentation rate (ESR) the ratio of blood to citrate is 4:1 (v/v). Although heparin can be used for routine haematological investigations. i isnot recommended because cells deteriorate more rapidly in heparinized than in EDTA- anticoagulated blood, and there is more distortion of leukocytes and platelets. “The inner wall of the capillary tubes may be coated with heparin 1 avoid blood cloutis investigation g prior to the laboratoryPreparation of blood films Microscopical examination of blood films identifies qualitative. morphological det blood cells that can serve ax indicators for specific disorders. Although the technique looks simple, the morphological examination is highly subjective and requires excellent experience on the part of the investigator to provide teliable data. This implies technical skill in the preparation of the material as well as sound knowledge of the identification of normal and abnormal blood cells and their typical morphological appearances. The microscopic examination should therefore be left to the most experienced investigator, who should also, supervise and train junior personnel during daily routine investigations. When venous blood is used for the preparation of the blood film it must be spread immediately alter collection, However, blood collected into EDTA and transported and/or stored at 20°C may be used up to one hour after collection for preparation of films for microscopic examination, Some parasites (Borrelia. trypanosomes and microfilariae) can be detected in a fresh wet blood film by their motility, but for species identification a permanent preparation may often be necessary. Two types of permanent blood films are used. cach providing different kinds of information. The thick film is mainly employed to increase the sensitivity of the test for detecting parasites in the blood, while the morphology of the parasites is best studied in the thin film (see also page 102). The accurate examination of blood films depends on the use of clean, grease-free slides. Old slides should first be cleaned in detergent. and washed with 70% alcohol. New slides should be cleaned with alcohol before use. Blood to be collected for the preparation of films should flow rely out of the vein. Blood that has been “milked” from the finger will be diluted with tissue fluid, thus decreasing the numbers of parasites per field Supplies Cotton balls Ethanol or isopropanol, 70%. Lancet, sterile Cover slip (wet film only) Normal saline (wet film only) Microscope slides, clean, grease-free and serateh-free Slides with perfectly smooth edges for the spreaders (thin film only) Fresh wet film Technique 1, Disinfect the ball of the third or fourth finger with alcohol and let the skin dry completely. With the lancet prick the lateral side of the ball, not too close to the nail bed.14 2. Place a first drop of blood, the size of a pinhead, on the centre of a slide, being carelul not to touch the skin. 3. Invert the slide and add an equal small drop of normal saline to the drop of blood. Mix the blood and saline with a corner of a cover slip. Cover the preparation with the cover slip, Press the slide with a thin stick or a match so that the centre of the blood film is almost colourless. Examine immediately. Thin blood film Technique 1, Making the spreaders: select a slide with perfectly smooth edges and make a diagonal seratch across the two comers at one end with a file and snap off a pair of pliers Disinfect the tip of the third or fourth finger with cthanol, With the lancet prick the lateral side of the ball, not too close to the nail bed. 3. Grasp the slide by its edge and from below; bring the end of the slide into contact with, a small drop of blood, being careful not to Iet the slide come into contact with the skin 4. Invert the slide and place it on a flat surface and steady it with the index finger and thumb of the left hand, if you are right-handed (if left-handed vice-versa). 5, Place the end of the spreader at an angle of 45° to the first slide slanting towards the drop of blood, Draw the spreader back until iC touches the drop of blood and wait until the blood has spread along the entire edge of the spreader. 6. With a firm fast motion push the spreader along the first slide maintaining the 45 angle (Fig. 3). In this way the blood is drawn afier the spreader in a thin smear which, if the original drop is small enough, ends in a drawn-out tail well before reaching the end of the first slide. 7. Wave the stide so it dries quickly. In humid seasons the drying of the film can he speeded up by waving the sfide $ cm away from the flame of spirit lamp, 8. With a lead pencil mark the thick part of the fitm with the patient's name or number he Iwo corners with o> ¢/ FIG.3 : Thin blood film15 Common faults in preparing thin blood films FAULT CAUSE The end of the film is lost The drop of the blood is too big The film ends in a thick line The spreader has been lifted up too early The end of the film is ragged The edge of the spreader is uneven Lines along the film Blood is clotting when the film is made Lines across the film The spreader was pushed forward jerkily Holes in the filrm Greasy slide Thick blood film Technique {. Disinfect the batt of the third or fourth finger with alcohol. With the lancet prick the lateral side of the ball, not too close to the nail bed, 2. Grasp the slide by its edge and from below bring the end of the slide into contact with a drop of blood. Be careful not to let the slide come into contact with the skin. 3. Invert the slide and place it on a flat surface and steady it with the index finger and the thumb of the left hand, if you are right-handed (if lelt-handed vice versa). 4, Spread the blood cvenly ina film ahout 10 mm wide with the corner of the slide, or a needle, or a toothpick, or by slowly rotating the slide (Fig. 4), The film should be spread quickly and have the correct thickness (one should be able to see the hands of a wrist-watch but not the figures through the smear). 5. Allow the film to dry (on a sunny bench, under a lamp or az electric fan), protected from dust and flies. The smear should be completely dry to make the blood film adhere to the slides. In the humid tropics prolonged drying may be necessary. Thick film should not be fixed. FIG.4 : Thick blood film16 Transport and storage of blood As a principal rule in laboratory investigation whole blood should not be stored. but immediately used for investigation, because blood cells are metabolizing and this causes changes in the composition of blood even after blood collection. Typically, blood glucose steadily decreases with storage. since it is metabolized by red blood corpuscles and is their only source of energy. Furthermore. blood, urine, other body fluids and excreta are excellent media for the growth of contaminating bacteria. Finally, certain analytes decay rapidly during storage (e.g, clotting factors VIII and V). If whole blood is stored for a few hours the concentration ot activities of a number of analytes will change. Depending on the temperature during storage, concentration may increase or decrease, and changes will be more pronounced as duration of storage increases. If blood, plasma or serum cannot be directly transported to the laboratory or immediately investigated. they should be kept in the dark at 4 to 8°C in stoppered vials to prevent evaporation of water and degradation of analytes by light In blood, plasma or serum the concentration or activity of an analyte will change with time (Table 5) depending upon storage conditions. The activity of some analytes will change within a short period of time. Serum stored for a long time is not recommended for electrophoresis. Results of determinations of triglycerides and cholesterol may be erroneous when using cooled. old, stored serum because of non-homogenicity of specimens. Routine investigation should therefore be made using fresh specimens, When samples need to be preserved for longer periods they should be frozen to ~18°C for storage up £9 one month and to -40°C or below for longer periods. TABLE 5: Analytes affected by prolonged storage of blood specimens Blood gases: K+ Chloride Phosphate Creatinine (determined by the Jaffé reaction) Glucose Serum iron Lactate dehydrogenase Alkaline phosphatase Aspartate aminotransferase Coagulation factors V, Vil, and VII! Erythrocytes Haemoglobin PCV (Haematocrit) White blood cells Reticulocytes Platelets Prothrombin time (PT) Activated partial thromboplastin time (APTT) Erythrocyte sedimentation rateTABLE 6: Stability of analytes in sterile serum stored in astoppered tube ANALYTE ¥C Bilirubin measurement in fresh serum only Chloride 10d 10d Creatinine 24h not recommended ron 7d kt 14d 14d Nat 14d 14d Phosphate 7d ed Protein 6d Triglyceride 2d not recommended Urea 3d 2ah Uric acid 5d 5d Alkaline phosphatase 7d 7d 10% decrease Cholinesterase 7d 7d Aspartate aminotransferase 3d 8% 3.d 10% decrease Alanine aminotransferase 3d 10% 3.17% decrease Lactate dehydrogenase 7d 7d It is therefore pertinent to separate plasma or serum from blood and provide appropriate storage conditions. This can be achieved by centrifugation of blood for 5 to 15 minutes al 1000 xg to 2000 xg. Plasma may be left in a separate tube for clotting to obtain serum, if requested. In glass tubes clotting proceeds faster than in plastic thes. where it may continue for hours if this reaction is not artificially accelerate, Plasma or serum should be separated avoiding exposure of the fluids to direct sunlight, which otherwise will cause degradation of bilirubin. Table 6 shows the stability of the commonest analytes measured in clinical chemistry after storage ina refrigerator and at ambient temperature. Haemoly: It is important to avoid haemolysis at every step during blood sampling, transportation and storage, because haemolysis causes specific or non-specific changes in measurements of a number of analytes. Artificial causes of haemolysis Blood sampling through a too small needle Forced suction of blood in the syringe during blood collection Vigorous shaking of blood in the syringe or test whe Forced expulsion of blood from the syringe. especially through a needle Centrifuging blood samples at high speed before completion of clottingFreezing and thawing of blood Unclean tubes with res Water (or hypotonic solutions) in syringe or tube al detergent Chemical tests affected by haemolysis Bilirubin Phosphate Serum iron Cholesterol Alkaline phosphatase Lavtate dehydrogenase Protein electrophoresis Sources of analytical variance after blood collection ‘Contamination (microbial and chemical) Prolonged transportation and storage prior to measurement duc to: — glucose uptake by blood cells — liberation of enzymes trom blood cells — liberation of ions from blood cells — rapid decay of enzyme activity Temperature — freezing: lipids and certain serum proteins — cooling: K+, LDH — heating: pH, many enzymes and coagulation factors Incorrect specimen identification Exposure to light: bilirubin Transport in open or poorly stoppered vials or leaving specimens uncapped on the bench, This leads to evaporation of water from plasma, or conversely water uptake in a humid atmosphere. Individual characteristics of samples, e.g. hyperlipaemia, hyperbilirubinaemia of paraproteinaemia in a patient. Collection and transport of blood for microbial investigations Blood cultures are important in clinical microbiology as they provide information on the severity and spread of the infcetion, they identify the aetiological agent, and allow isolation of the organism for susceptibitity testing. Investigations have shown that between 5% and 30% of positive blood cultures represent contamination with skin bacteria. Both Gram positive and Gram negative bacteria are found on the skin of healthy persons, but normally the Gram negatives are found in low numbers. in hospital patients and in patients receiving antibiotics the Gram negative bacteria increase considerably in numbers. Though many of these contaminants can be recognized as19 commensals om the basis of their identity, some are very difficult to recognize. Therefore, to keep the numbers of contaminants low, the use of a proper skin antisepsis is extremely important for the colicction of blood for hacterial culture. Recommended agents for skin antisepsis prior to blood sampling ‘There are several alternatives for skin antisepsis, but the following are recommended for blood culture: © Tincture of iodine 1% or 2% is ive and convenient as one can visually per the area of skin which has beer disinfected. However, some patients have iodine hypersensitivity and jt is recommended that the iodine he removed with alcohol after hlood collection © 10% povidone-iodine (iso-betadine) causes less irritation to the skin but has the disadvantage that the disinfected area is not easily perceived visually © 0.5% chlorhexidine (hihitane) in 70% alcohol causes less irritation and sensitivity to the skin hut has the same disadvantage as povidone-iodine, All three solutions should be kept in airtight bottles as evaporation of the alcohol substantially reduces the activity and efficacy of the antiseptic. Venipuncture Selection of the venipuncture site ‘The site of blood collection influences the number of contaminants. Blood drawn directly from the antecubital vein usually has the lowest number of contaminants, while higher counts: are found when blood is drawn from the ferhoral vein and much higher ones when blood is drawn trom the umbilical vein in newborns, High contamination rates are alse found in blood drawn from intravenous catheters left in place for more than 48 hours. Preparation of the venipuncture site and containers No antiseptic works instantly: a contact time of at least I-2 niinutes is required. A further reduction of the skin flora is achieved if the antiseptic is well rubbed on to the skin. The rubber diaphragm of the blvod culture bottle (unless protected) is potentially contaminated and should be properly disinfected before the blood is injected. The same antiseptics as used for the skin can be used for this purpose Blood cultures Number of blood cultures The number of blood cultures necessary for detecting bacteraemia depends theoretically on the volume of blood inoculated (Table 7), the time of collection, the type of infection and the infecting organism, thé age of the patient, and the treatment of the patient with antibiotics prior20 to collection. The number of bacteria in the blood is generally higher in the acute. initial stage of the disease than at a later stage, and small children usually have higher numbers of bacteria in the blood than adults. The number of bacteria is higher at high temperature of the patient with fever. For patients expected to seed bacteria intermittently into the blood, 80% of bacteraemias are detected with the first culture and 99% with the first three cultures. More than three cultures are therefore not necessary, unless the patient has received antibiotics. In this case, a new scries of blood cultures may be indicated when the antibiotics have been stopped. Lf time permits it is recommended that the blood cultures be taken at intervals of one hour. If this is not possible due to the severity of the illness, they should be drawn from two different sites. TABLE 7; Total volume inoculated in sets of two culture bottles for aerobic and anaerobic cultivation AGE VOLUME Children below 2 years 2 mL in one set of bottles Children 2-5 years 8 mL in two sets of bottles Children 6-10 years 42 mL in two sets of bottles Children 11-15 years 20 mL in two sets of bottles Children above 15 years and adults 30 mL in three sets of bottles Procedure for blood culturing Supplies Blood culture bottles (sets of two) Antiseptic solution (Lincture of iodine. povidone-iodine or chlorhexidinc-alcohol) Cotton balls Needles, bore size 19 or 20 Syringes, 10 mL Tourniquet Technique 1. Explain to the patient what will happen. Assemble and prepare the equipment: remove the protective cap from the culture bottles, if present. . Apply tourniquet and select an appropriate vein for the collection, usually the median elbow vein. 3. Prepare the site with antiseptic: apply the antiseptic and rub a 5 cm? area around the selected site for | minute. Leave the antiseptic to dry and discard the swab. Make a new swab and cleanse the site again, beginning at the centre and scrubbing in a circular motion outwards. Let it dry. 4. Disinfect the diaphragm of the blood culture bottles as described for the skin. If it is necessary to palpate the site before venipuncture, disinfect the finger with the same2 procedure as described for the skin of the patient. Do not touch the prepared site with fingers that are not disinfected 5. Place the needle on the syringe into the vein, and draw the volume needed for the culture. 6. Remove the tourniquet, withdraw the needle rom the vein, immediately apply pressure to the puncture site with a clean cotton ball and bend the elbow. 7. Snocatate the culture bottles carefully adding the correct amount of blood to each bottle. Be careful that no air is injected. 8. Put labels on the bottles $0 as not to cover the area occupied by the medium, Indicate patient identification, ward number, and time of collection. 9. Bring the blood culture bottles immediately to the laboratory, where the bottles are placed directly in the incubator, Do not store inoculated bottles in the refrigerator Selection of blood culture media Comparison of different blood culture media has shown that for isolation of aerobic. facultative and anaerobic bacteria tryptic soya casein broth (TSB) medium is as good as, and with some bacteria even superior to, other media. This makes tryptic soya casein broth the medium of choice for both aerobic and anaerobic culture, Pleural flu’ Abnormal accumulation of cxudate or transudate may occur in the pleural cavity or peritoneal space of patients with certain diseases. These fluids can be aspirated for therapeutic and/or diagnostic reasons. Supplies Antiseptic solution (e.g. tincture of iodine) © Syringe 10 mL © Needle, sterile, with bore size 19 or 20 © Specimen container Technique 1. Identify the presence and extension of pleural fuid by clinical inspection of the patient and palpation of his/her thorax and/or abdomen on the patient's back and lateral side. 2. Ask the patient to sit on a chair facing towards the back of the chair. Let the patient put his/her arms on the back of the chair and beri so that the spine is curved forward. 3. Locate the intercastal space between the 8th and 9th ribs by counting the number of ribs close to the spine, starting, from the neck. 4. Cleanse the intercostal space between these ribs about 12 em to 15 em lateral to the spine cither on the left or right side of the patient's body, depending on the focation of the pleural fluid, with 70% aleohol, 2% tincture of iodine, povidone-iodine or 0.5% chlorhexidine-alcohol.22 5. Take a 10 mL syringe (for the purpose of diagnostic investigation of the fluid). connect the syringe to a sterile needle, and puncture with the syringe at right angles to the skin just above the upper edge of the lower rib (if the puncture is made at the lower edge of the rib the intercostal nerve of the rib may be damaged). Then push the needle through the skin until pleural fluid can be aspirated by slow suction with the plunger of the syringe. 6. The filled syringe and needle are removed by rapid extraction and the small wound covered with a stcrile gauze pad. Sometimes pleural fluid cannot be aspirated although its presence is indicated by clinical or radiological exarnination. In this case the next lower intercostal space may be selected for the puncture. 7. Indicate the appearance of the pleural fluid on the request form as follows: © Bloody ~ indicating either traumatic puncture or internal bleeding. e.g. from an injury or spontaneous rupture. ¢ Turbid — indicating a fibrin deposit or a high cell content from an infection or a malignant disease ¢ Milky ~ indicating seepage of the lymphatic system. Pleural fluid in the absence of these features is transparent and yellowish Peritoneal fluid Supplies The supplies for aspiration of the peritoneal !uid are the same as for aspiration of pleural fluid. Technique 1. Let the patient be in a supine position on a bed or stretcher and by palpation and percussion locate the extent of the peritoneal fluid, Locate the kidney. the liver and spleen to avoid accidental injury te these organs. 2. Cleanse the skin at the site of puncture twice with antiseptic (e.g. tincture of iodine). letting it dry afler each pplication. 3, Assemble the syringe and needle. Push the needle through the skin and by making a slight vacuum with the sycinge plunger continue to push the needle forward. until peritoneal fluid is aspirated 4. When the syringe is filled, withdraw the needle quickly and cover the puncture wound with a dry sterile gauze pad. 5. Indicate the appearance of the peritoneal fluid on the request form, as follows: Bloody ~ indicating cither a traumatic puncture or internal bleeding. e.g. from an injury or spontaneous rupture. © Turbid — indicating a fibrin deposit or a high cell content from an infection or a malignant disease © Milky — indicating seepage from the lymphatic system. ‘© Greenish ~ indicating efflux of bile. Peritoneal fluid in the absence of these characteristics is yellowish and transparent.23 6. The aspirate should be sent, without delay, to the laboratory for microscopic and chemical investigations as well as microbiological culture. Cerebrospinal fluid The cerebrospinal fluid (CSF) is a body fluid that surrounds the spinal cord and the brain. The compartment of the CSF is separated from the hlood system by a barrier, the blood-liquor barrier. This barrier prevents blood cells and the majority of plasma proteins from entering into the CSF while water-soluble small molecules, such as glucose, penetrate the barrier, Normal CSF is therefore almost void of cells and appears colourless and trnsparent. Depending on the localization of CSF collection, CSF protein concentration may differ (Table 8). These concentrations are also related to the age of the patient (Table 9). TABLE 8: Reference ranges of CSF protein concentration in adults IT! INCENTRATIO! Ventricular 0.05-0.15 g/L Cisternal 0.15-0.25 g/L Lumbar 0.15-0.45 g/L TABLE 9: CSF protein concentration by age (from lumbar puncture) AGE, CONCENTRATION Neonate 0.25-0.72 giL 1-6 months 0.15-0.72 gL 6-12 months 0.15-0.50 g/L 1-48 years 0.10-0.43 g/L Adults 0.15-0.45 g/L an essential step in the diagnosis of any patient with evidence of | irritation or affected cerebrum. CSF is collected for the diagnosis of infectious (viral, bacterial, fungal and parasitic) diseases, of intracranial haemorrhage. of malignant disease and of disturbances of the blood-brain barrier. CSF is not collected trom patients with clinical signs of intracranial pressure. Some analytes (e.g. albumin, immunoglobulin} are measured in the CSF and in serum for the differential diagnosis of malignant or infectious diseases.24 ‘The description of the method of collection of CSF specimens is beyond the scope of this Manual, since this should be done only by a physician or a specialty trained nurse. At Icast two separate tubes should be collected, each containing 3-10 mL of CSF. One tube is forwarded for chemical and microscopic analysis, the other for microbiological investigation. The CSF is cultured under acrobic conditions. Anaerobic culture should be performed if one of the following conditions is suspected (and should be mentioned on the request form): brain abscess, and middle ear, mastoid of sinus infections, CSF shunt, head trauma or craniotomy. Remember © De not delay transport and laboratory investigation. Once the CSF is removed, cells and parasites are rapidly lysed: also glucose is rapidly metabolized by cells and bacteria in the CSP. Therefore CSF must be transported in a refrigerated hox if a delay before processing cannot be avoided. © Work carefully and economically during laboratory investigation since CSF collection is difficult and quite often cannot be repeated. ‘¢ Physical inspection (colour. turbidity) and Pandy's reaction should be perlormed, using an aliquot volume of the collected CSF, immediately after puncture at patient's side. Interpretation of microscopic and chemical investigation CSF from a patient may have the following appearance: Clear and colourless — normal CSF Clear with Tyndall effect (= sparkling appearance against incident light) — elevated concentration of protein Clear yellowish (= xanthochrome) — sign of old hae! Clear red — sign of acute hemolysis of RBCs Turbid blood-stained — hacmorthage Turbid white — high cell of protein content Turbid clots (after storage for 12 to 24 hours) — fibrin clots. jolysis of RBCs weeoe Normal CSF has the same pH as serum, namely 7.4. The concentration of sodium is 140 mmol/L, potassium 2-2.5 mmol/L, and bicarhonate 23 mmol/L. The glucose content is usually about 60% of the blood glucose, and varies with the blood-glucose level. It is raised in uncontrolled diabetes and is often high in uraemia. The protein content is normally below 0.45 g/L. Protein may sometimes be slightly increased in diseases of the brain. Very high levels are found in spinal block. e.g, tumours in the spinal canal. polyradiculitis. subarachnoid haemorrhage, bacterial, fungal (Cryptococeus) and parasitic (Trypanosoma) infections. A bloodstained CSF may be caused by a subarachnoid haemorrhage or by an accidental puncture of an intraspinal vein hy the needle. In the latter case the number of erythrocytes is greater in the first few millilitres during collection. Also the supernatant of the CSF will be colourless after centrifugation. In case of subarachnoidal haemorrhage the CSF will be uniformly stained and the supernatant fluid will often be slightly yellow (xanthochrome). Low25 concentrations of erythrocytes will easily haemolyse and also cause xanthochromia, which may lead to a false interpretation of the findings. The CSF contains a few lymphocytes (less than 5 x 10° per litre). A slight non-specific increase of lymphocytes may be found in many diseases affecting the cerebrospinal system. In patients with meningitis the cells of the CSF are always elevated, although the number may vary between (07 and 10!! per litre. Total and differential white cell count is essential, particularly in the differentiation of bacterial and non-bacterial meningitis. In bacterial meningitis, particularly the pneumococcal and staphylococcal forms, granulocyte count may sometimes be low in the early stages of the infection, but frequently the leukocyte count is high. In the initial phase of bacterial meningitis, polymorphonuclear leukocytes predominate, but as recovery takes place, these are gradually replaced by lymphocytes. Polymorphonuclear leukocytes may also be present, along with lymphocytes, in tuberculous meningitis, cerebral abscess, and in the early stages of poliomyelitis. A predominance of lymphocytes is the typical picture in viral meningitis and encephalitis, active neurosyphilis, tuberculous meningitis and in late stages of bacterial infection. Eosinophilic pleocytosis is seen in certain helminthic infections of the central nervous system (angiostrongyliasis, gnathostomiasis, paragonimiasis and ncurocysticercosis). In bacterial meningitis the glucose level is usually low both is usually normal in non-bacterial meningitis. In partially treated bacterial meningitis. ¢ in a patient who has received antibiotic therapy, the differentiation between bacterial and viral meningitis by microscopy can be difficult, Recently it was found in children that measurement of activity of phosphohexose isomerase (PHD, an enzyme predominantly produced by polymorphonuclear cells, allows good discrimination between viral and bacterial infection from CSF with borderline cell counts. Low glucose concentrations should be taken as an indication for intravenous antibiotic therapy. ind protein is high: however. one or In untreated bacterial meningitis. the aetiology can be identified by Gram’s stain preparation with a probability of 80% approximately The presence of fibrin clots in the CSF indicates an inflammatory process by which fibrinogen can enter the spinal fluid. This feature develops frequently in tuberculous meningitis. Throat swabs Examination of throat and nasopharyngeal specimens has a triple purpose. It confirms the diagnosis of streptococcal pharyngitis, diphtheria, epiglottitis, Vincent’s stomatitis and oral candidiasis (thrush); it may establish the focus of infection of scarlet fever, rheumatic fever and acute glomerulonephritis. It may also identify carriers of diphtheria bacilli, gonococei and meningococci. The list of organisms which can cause sore throat is long: diagnosis of diphtheria in a patient should be made as soon as possible. Good history taking, inspection of the throat in a good light and examination of a film will in many cases allow for a preliminary diagnosis of diphtheria, but a final diagnosis will depend on the outcome of the culture of the throat swab,26 Similar investigations will also serve to determine if any of the other organisms mentioned above is fesponsible. Commonly, throat swabs are only cultured for Streptococcus pyogens and (in endemi countries) for Corynebacterium diphtheriae. Pneumococci, Haemophilus influen-ae. Staphylococcus aureus or enteric bacilli will not cause sore theoat and, if isolated, should not be reported. If other infections are suspected this should be stated on the request form, Oral candidiasis or thrush appears as greyish-white membranes loosely adherent to the oral mucosa of the inner cheek, palate, tonsillar pillar and pharynx, It is mostly seen in neonates and infants, diabetics, and patients receiving broad-spectrum antibiotics or corticosteroids. Material for microscopy and culture is taken from the membranes, using the same technique described below for throat cultures. Vincent's stomatitis is a pseudomembranous or ulcerative infection of the gums, cheeks, and tonsils. It is generally thought to be caused by a Gram-negative spirochacte, Treponema vincentii, and a spindle-shaped anaerobic rod, Fusohacterinm nucieatum, The pseudomembranes on the tonsils may be mistaken for diphtheria, but can be easily differentiated by examination of a Giemsa-stained film of the membrane. Cultures are of no value for the diagnosis, but should be done to rule out diphtheris Epiglottitis is an uncommon but sometimes fatal inflammatory oedema of the epiglottis and surrounding mucosa, usually associated with Haemophilus influenzae. type b. However. the pharynx should not be swabbed unless an urgent trachcostomy can be performed, as this may cause a reflex Jaryngospasin and further obstruct the airway. A milder but superficially similar disease due 10 inflammatory oedema of the supraglottic area can be scen in some virus infections. Whooping cough (pertussis) is an acute inflammatory reaction of the trachea and bronchi due to Bordetella pertussis. The typical case starts as a common cold with an irritating cough which after one or two weeks becomes paroxysmal. In infancy there may be no typical cough but the patient may present only with spells of cyanosis and apnoea, and older children and adults may have only protracted cough. In most instances there is no need for bacteriological examination. Nasopharyngeal cultures are recommended when attempting to isolate the organism. The highest incidence of recovery of Bordetella pertussis is in the initial catarrhal stage; when the paroxysms start, the incidence falls and in the fourth week the organism can only be isolated in 40% of eases. Supplies © Cotton swab © Transport medium (Stuart's, Amies and Cary Blair) Tongue depressor Technique 1, Whenever possible the specimen should be obtained before administration of antimicrobial therapy.27 Nv Focus the light so that the oral cavity is well illuminated and ask the patient to open the mouth widely and breathe deeply. 3. Depress the tongue gently with a tongue depressor. Ask the patient to say “ah" while a swab is carefully introduced over the tongue into the oropharynx. Care should be taken to avoid touching the lips, palate or the tongue with the swab. 4, Rub the swab firmly over the back of the throat, both tonsils and any areas of inflammation, exudation, or ulceration. A. separate swab should be taken for microscopy if diphtheria, candidiasis or Vincent’s angina is suspected. 5. If the swab is to be processed within 1-2 hours, place it in a sterile test tube and close with stopper. If the processing will be delayed, place the swah in transport medium. Remove the stopper without touching the sterile end, insert the swab into the transport medium, break off the non-sterile part of the swab, which has been in contact with the fingers, and replace the stopper. Interpretation The diagnosis of diphtheria is primarily based on the clinical examination. Although the appearance of diphtheria bacilli in stained films from membranes is highly characteristic, these organisms cannot be identified by their morphology alone, since they may be indistinguishable from other non-pathogenic corynebacteria. The diagnosis can therefore only be presumptive. The final identification is obtained only after isolation of toxigenic C. diphtheriae {rom the patient; this will require several days of microbiological investigation. Isolation of Sireprococcus pyogenes should establish the diagnosis of streptococcal sore throat; but the organism should be found fairly abundant in culture if it is to be regarded as causal and not merely a casual infeetion (carrier). Sputum A sputum specimen is sent co the laboratory for the actiological investigation of bacterial and fungal infections of the deep respiratory tract including the bronchial tree and the Lung parenchyma. In some countries sputum can also he used for the microscopic detection of bronchopulmonary parasites: ova of Paragoninius spp. and larvac of Strongyloides stercoralis. In many laboratories expectorated sputum is the most frequent type of specimen submitted for microbiological diagnosis. However, with the exception of the diagnosis of tuberculosis. microbiologieal investigation is often useless for the correct treatment of the patient. It may even give misleading results and lead to unnecessary treatment with broad spectrum antibiotics. All expectorated sputum is contaminated 10 some degree with secretions of the oropharyngeal cavity, which contain a wide variety of commensal bacteria. Some of these are potential pathogens of the lower respiratory tract (pncumococei, Haemophilus influenzae). Contamination with oropharyngeal secretions should be minimal, since the sputum reflects the infectious process in the bronchi and the lungs. This requires a good technique for expectoration and collection of the specimen.28 Supplies ¢ Specimen container, sterile, wide-mouthed Technique 1. Whenever possible the specimen should be collected prior to antimicrobial therapy. Thorough instructions should be given to the patient by a nurse or laboratory technician. Sputum should preferably be collected in the morning. The patient should be standing. if possible, or sitting upright in bed. 3. The patient should take a very deep breath to fill the lungs with air and expirate in one breath, coughing as hard and deeply as possible. 4. The sputum which is coughed up should be spat into the container. 5.If the quantity of the sputum is not sufficient, the procedure may be repeated. A well-collected specimen on a single occasion, however. is better than a speciinen collected over several hours. A 24-hour collection of sputum or repeated (three) specimens are useful only for the diagnosis of mycobacterial disease. 6, Macroscopic evaluation of sputum should be made. Before sending the specimen to the laboratory, it must be briefly inspected and its appearance should be recorded on the request form, as follows — a watery, white-frothy. or mucoid sputum generally represents pharyngeal secretions and should not be examined for non-tuberculous infections: — a purulent, mucopurulent (mixture of pus and purulent flecks), yellow. green of brown blood-stained sputum is generally acceptable. 7. Transport. After collection of the sputum, the cap of the container must be tightened. and the specimen must be sent immediately to the laboratory. In case of delay, the specimen should be kept in the refrigerator. Microscopie screening of the sputum. based on the evaluation of the proportion of leukocytes versus epithelial cells, should be done immediately after collection. w When the patient does not expectorate, as is often the case in children, tracheal secretion can be aspirated through @ nasopharyngeal catheter. The culture results, however, are difficult to interpret in children because of the presence of potential pathogens (pneumococci and Haemophilus influenzae) in the normal pharyngeal flora. For severely ill hospitalized patients who are suspected to. be suffering from non-tuberculous bronchopulmonary infection, special techniques are available for colleesing deep respiratory tract secretions with no or minimal contamination by the oropharyngeal flora: © transtracheal aspiration * bronchoscopic aspiration or brushing bronchoalveolar lavage direct needle aspiration from the lung parenchyma, ‘As these procedures require special equipment, or may be associated with serious complications, they should not be used at the primary health care level.A blood culture should be made in all seriously ill hospitalized patients with a clinical diagnosis of pneumonia, since blood culture is positive in 20-30% of patients with bacterial pneumonia. Gastric washing or aspirates are processed only for mycobacteria as other organisms ate rapidly killed by the high acidity of the stomach content, Urine Urine is collected for chemical, cytological and microbiological investigations. In healthy persons the urine in the kidney and the bladder is sterile. The lower parts of the usethra and the genitalia are normally colonized by bacteria, many of which can also cause urinary tract infection. This is why even the most carefully collected urine sample will be contaminated. A urinary (ract infection can be predicted from collecting a midstream urine sample in a sterile container and counting the number of bacteria per millilitre. A bacterial count of > 10° bacteria/mL in a midstream sample of urine will indicate a urinary tract infection in over 80% of cases. About 30% of patients with urine having 104 ( 105 bacteria/mL, and only 4% of patients with urine counts of <{G* bacteria/mL, have a urinary tract infection. The significance of fower counts increases when associated with pyuria A random urine specimen collected in a clean, but non-sterile container ix used for qualitative or semi-quantitative examination of contents such as glucose, protein, pH, specific gravity, bile pigments, and possible presence of blood. pus, or crystals, Often the first iiorning-voided specimen is requested because it gives the urine concentration most accurately, Otherwise the time of collection or volume of the voiding is not important. Quantitative urine analysis is helpful for assessment of kidney function For chemical and microbielogical examination the urine must be collected “clean”, discharge or pus from the vagina or external genitals added to the urine will invalidate the examination. For the demonstration of eggs of schistosomes a random urine sample should preferably be collected between 10 am and 2 pm, as the concentrations of eggs are greater during this period. particularly in the Jast drops of the passed urine. Exercise prior to the collection will result in an excretion of more eggs, Urine collection Patients not requiring assistance I. Give the patient a clean, preferably sterile container of appropriate size (50 mL. or more: for quantitative chemical investigations the whole volume excreted during 24 hours must be carefully collected in a 2-litre container). The container must be free of detergents, which may cause false determinations. The container should be pre-labelled with identification data, at least the patient's first name and surname. 2, Instruct the patient before the collection, preferably with Hustrations. Tell him or her ot to touch the inside or rizw of the container. Ensure that his/her hands are clean.3. Male — If not circumcised, withdraw the foreskin. — Let the patient begin to urinate, but passing the first portion to the toilet. — Collect the mid-stream sample of urine in the container and pass the remainder into the toilet, Female —— Instruct the patient to squat over the toilet and separate the labia minora with one hand. — The patient should void the first portion of urine into the toilet, while with her hand, keeping labia separated. — The mid-stream sample of urine should be collected in the clean sterile container. without contaminating the lip or inside of the container with the hand. or inguinal or perineal area. The remainder should be passed into the toilet. — The container is capped without holding its top. Bedridden patients requiring assistance 1. Prepare a clean, sterile container of appropriate size, cotion balls, gauze pads, soapy water and bed pan. 2. Explain to the patient how urine will be collected, 3. Male — Withdraw the foreskin and clean the glans with cotton balls soaked with soapy water, While working away from the urethra. — Ask the patient to urinate into the bed-pan. — Collect the rnid-stream sample of urine in the specimen container. Female — Place the patient in lithotomy position. — Wearing sierile gloves. separate the labia minora with one hand and carctully clean around the urethra with cotton balls soaked with soupy water while wiping from front 10 back. — Rinse the cleansed area with two successive sterile. water-soaked cotton balls, wiping from front to back. — Ask the patient to urinate into the bed-pan — Collect the mid-siream sample of urine in the appropriate container without contaminating the container (“clean catch"). —_ Any excess urine is passed into the bed-pan. Infants 1, Prepare a clean, soapy water, 2. Let the child drink as much water as possible just prior to the urine collection. 3. Clean the external genitalia as described above. 4, Seat the child on the lap of the mother, nurse or ward attendant. 5, Collect as much urine as possible in the container from the urinating child. terile plastic bag of appropriate size, cotton balls, gauze pads and31 Note: For quantitative chemical investigation the child is not allowed © drink prior to urination, and special attention is required for the collection of the total volume excreted over 12 oF 24 hours. Transportation of urine Urine specimens should be transported to the laboratory within one hour for chemical and microbiological investigations, the reason being the growth of bacteria. A specimen containing 103 bacteria/mL after collection may have 105 bacteria/mL 2 hours later when kept at ambient temperature. If transport carmot be immediately assured, the specimen should be refrigerated and processed within 24 hours, Additives are not required. Sexually transmitted disease specimens Sexually transmitted diseases (STD) are caused by a large variety of viruses, bacteria, fungi and parasites. With a few exceptions all these microorganisms are too delicate and fastidious to grow in vitro. Their identification depends therefore upon the collection of an appropriate specimen and upon its transportation under optimal conditions to the laboratory, As the laboratory diagnosis of Chlamydia trachomatis and herpes simplex virus is generally not possible at the primary or intermediate health care level, specimen management for those organisms will not be considered here. All specimens should be collected before the administration of antibiotics or the apptication of topical drugs. Gonorrhoea Gonorrhoca should never be diagnosed without the aid of microscopy or culture. in the majority of males the diagnosis of acute gonorrhoea can he easily made. A microscopic examination of a Gram-stained urethral smezr will, in 80-90% of cases, Iead 10 a correct diagnosis. Only in asymptomatic patients the diagnosis may be missed by microscopic examination and cultural verification will then be essential In females. acute gonorrhoca usually involves the endocervical canal, the urethra. and the Bartholin's and Skene’s glands. When symptoms are present, the case will be referred to specialists. However, in the majority of females there will be no ar only slight non-specific symptoms, and culture examination becomes muct more important for a correct diagnosis. Gonecoccal urethritis in males In case of abundant exudate, a drop of the purulent discharge is collected with a sterile swab or loop and immediately used to prepare a thin film on a clean slide and to inocatate the culture plate. If no discharge is evident, the urethra is stripped towards the orifice to express some pus and a thin sterile swab is inserted 2-3 cm into the urethra and rotated before being withdrawn. If delay in the inoculation of the cultures cannot be avoided, the swab must be inserted into a transport medium (Stuart or preferably Amies), Gonococei will remain viable for up to 24 hours at room temperature under sueh conditions, Investigation of urine sedimentis not recommended for the diagnosis of gonococcal urethritis, although it may be used for the microscopic detection of Trichomonas vaginalis, Prostatic massage does not increase the recovery of gonococci from the urethra. Gonococcal cervicitis in females The endocervical canal is the primary site for the diagnosis of female gonorrhoea. For collection of exudate, a speculum moistened with warm tap water should be used. A sterile swah is inserted 2-3 cm into the cervical canal and rotated for a few seconds to permit absorption of the exudate. The swab should be immediately inoculated on to appropriate culture plates or sent to the laboratory in a transport medium (see above). The swab can also he used for the preparation of a Gram-stained smear, but the low sensitivity and specificity of microscopic diagnosis should be kept in mind, Vaginal discharge Increased vaginal discharge may reflect vaginitis caused by Candida atbicans (or other Candida spp.) or by Trichomonas vaginalis, Ik may also correspond to bacterial vaginosis, a syndrome due to the synergistic action of Garilnerella vaginalis and some strictly anaerobic bacteria. Vaginal fluid should be collected from the posterior fornix using a speculum and a cotton tipped swab. The exudate is immediately mixed with a drop of saline on a slide, covered with a cover slip, and examined under the dry x40 objective for motile Trichomonas, budding yeasts with pseudohyphae, or clue ceils studded with Gardnerella, Yeasts can be easily visualized by adding a drop of 10% potassium hydroxide (KOH) to the exudate, Culture of these organisms will also detect a number of asymptomatic carriers, and is thetefore not recommended Genital ulcers Genital ulcers should be examined for Treponema palliduny and for Haemophilus ducreyi. Dark field microscopy is recommended for the immediate detection of Treponema pallidum After wiping off the surface of the lesion with a dry gauze pad, serous fluid is expressed from the ulcer base. transferred with a wire loop on to a clean slide, covered with a cover slip and immediately examined. Material for cultivation of H. ducreyi should be sampled from the ulcer base using a swab, and immediately used to inoculate a selective culture plate. A Gram-stained smear is not reliable for the diagnosis of 1. ducreyi and transport of the specimen at distance is not possible Blood for serology A blood specimen (minimum 5 mL) for the serological diagnosis of syphilis or HIV infection should be collected, stored, und transported in a clean dry tube, as for other serological tests. If the result of the first specimen is negative in a patient with suspected primary chancre, a second blood specimen should be tested after 3-4 weeks.33 Stools Stool specimens are submitted to the laboratory for a number of investigations, which must be clearly indicated on the request form, as follows: chemical analysis (e.g. occult blood), bacteriological analysis (stool culture), detection of virus particles or antigens (e.g. rotavirus), or the microscopic diagnosis of intestinal parasites. As a general rule the same specimen can be used for several examinations. For the diagnosis of pinworm (Enterobius vermicularis) infection, special techniques must be used (e.g. cellophane tape). Faecal specimens for the aetiological diagnosis of acute infectious diarthoea should be collected in the early stage of illness and prior to treatment with antimicrobials. A stool specimen rather than a rectal swab is preferred, particularly for parasitological examinations. Except for some protozoan (Giardia and Entamoeba histolytica) infections and Strongyloides stercoralis infections, more than one specimen is seldom required for a correct diagnosis. Technique of stool collection 1. Faceal material should be collected directly in the container or clean bed-pan, or on a paper towel or piece of toilet tissue. 2. The material should then be transferred to a suitable container provided with a lid: a clean glass cup, a plastic or waxed cardboard box, or a special container with a spoon attached to the stop or the fid, The specimen should contain at least 5 g for parasitological and bacteriological examinations and 50 g for chemical analysis, Parts that contain blood and/or mucus, when present, should be selected for parasitological and bactetiological analysis. The specimen should not be contaminated with urine Stool specimens must not be left exposed to the air in containers without lids. Penicillin bottles, match boxes and banana leaves should not be used as containers as they expose the nursing and the laboratory staff to the risk of infection. 3. Rectal swabs should only be taken if the patient is unable to produce a stool specimen. A cotton-tipped swab. which need not necessarily be sterile, is introduced past the anal sphincter, rotated and withdraw, and inserted into a tightly closed tube. If the swab is not immediately processed it must be placed in a suitable transport medium (see below), the stick broken off, and the tube or bottle tightly closed before transportation 4. The clinician, the nurse or the technician should inspect every stool specimen and record the observations on the request form. Note the consistency (watery. liquid, mushy, formed) and the presence of blood, pus or mucus. In rare instances, aduit parasitic worms can be recognized with the naked eye: Ascaris, Enterobins, and tapeworm segments. 5. Storage and transport of specimens. Specimens for bacteriological examination should be transported to the laboratory and processed within a few hours. In case of delay the specimen should be refrigerated. If longer delays cannot be avoided a special transport medium should be used. In such media pathogens survive for up to one week even at room temperature, although refrigeration is preferahle © Cary-Blair transport medium is suitable for the conservation of ali enteric pathogens. It is commercially available as dehydrated powder and can be prepared and stored in small screw-capped glass containers (¢.g. Bijou bottles). The faecal specimen should6. be repeatedly inoculsted with an applicator stick all the way to the bottom of the transport medium, In the case of a rectal swab, a portion of the stick should be broken off before closing the container. Cary-Blair medium may be replaced by Stuart or Amies transport media. Buffered glycerol-saline solution prevents intestinal flora from overgrowing the enteric bacilli. It is not suitable for the conservation of Vibrio and Campylobacter. A solution is easy (0 prepare from its ingredients and is distributed in screw-capped wide-mouthed bottles (1 ounce), Alkaline peptone water is an excellent transport and enrichment medium for Vibrio cholerae and other Vibrio spp. Stool for parasitological examination can be preserved for several weeks by mixing the specimen with at least 3 volumes of preservative fluid. A 10% formalin 3.4% formaldehyde) solution is recommended and is prepared by diluting SO mL. of commercial formalin (34%) with 450 mL of distilled water. For the detection of motile forms of Entamocha histolytica and other protozoa, suspected stools should be examined within one hour afier defecation, without preliminary refrigeration, t is generally recommended 10 perform the examination immediately on the spot. A wel mount in saline of (reshly passed faeces is prepared and examined al once under the microscope Microscopic examination af stools for amoebae requires technical expertise. False results are Common, especially when examination is done by a non-expert. It is advisable 10 preserve specimens of fresh stools in formalin (for cysts) and polyvinyl alcohol (for trophozoites and cysts) to be examined by the nearest expert laboratory . If stool specimens are shipped through the mail, the local postal requirements must be respected, The leak-proof specimen container must be enclosed in a strong outer container which has sufficient absorbent material {0 absorb liquid in case of breakage of the specimen container.2. CONTROL OF LABORATORY INVESTIGATIONS Both the general aspects of internal quality contro! in a laboratory and the technical aspects need (0 be taken into account for the determination of each analyte relating to the method of investigation. All the methods used have their limitations: these may include simplicity or complexity of the method, the equipment used, analytical sensitivity, specificity or availability of reagents or the cost of investigation. For each analyte a number of methods or modifications of a method has been developed. Several criteria should be kept in mind when deciding upon whether a specimen can be further processed for laboratory investigation or must be rejected. Such a decision must be made in the light of the specific requested investigations. Laboratory investigation of a sample is a waste of time and money if certain criteria are not fulfilled. CRITERIA FOR REJECTION OF SPECIMENS Missing or inadequate identification Insufficient volume Specimen collected in wrong collection tube Contamination Inappropriate transport and storage Unknown time delay Errors may occur at any time and at each step in the overal! process of investigations, even in the best equipped and experienced laboratory. The application of certain practical rules is helpful to reduce such errors in daily work, to avoid misinterpretation of results and/or to identify quickly reasons in case of evident failure. The application of such rules facilitates the management of a laboratory and helps to economize on cost and time. Some of the measures are common to most, if not all, laboratory investigations; others are specific to an individual method. For didactic reasons, general measures and specific measures are discussed separately. Their application is suitable to health laboratories at all levels. Terms used to describe a general measure are explained below.General measures Calibration Quantitative measurement procedures are based on calibration. Calibration materials (calibrators) are reference materials which are used exclusively for calibration purposes. They must not be used for controlling analytical procedures; control materials, such as control sera, should not be used for calibrating methods or test systems. Solutions of calibration materials, should never be used for supervising the analytical procedures in quality control. The procedures of calibrating an analytical method have to be described in detail in a laboratory manual (see page 40). When introducing a new method or training new laboratory staff, solutions of calibrating material should be measured in dilution series of 5 to 10 different concentrations. The "calibration curve” obtained in this manner is used for control of linearity, limits of detection and working range within lower and upper limits. In routine work, usually a "one point calibration” may be satisfactory. using a concentration of calibrator at the upper range of expected values. For some analytes, v.g. protein or haemoglobin, daily calibration should be monitored by means of a control chart, similar to the quality control charts for accuracy. Sclf-calibrating instrument devices should be checked by manual calibration at feast once a month. Duplicate tests on a patient's specimen This is the casiest procedure to carry out; if there are too many test requests for measurements in duplicate, test at ‘east 10 consceutive specimens in duplicate, then calculate the standard deviation (SD). None of the duplicate tests should differ from one another by more than twice the calculated SD. The SD will be large and will not be sensitive to individual errors if the method of measurement is not under control. Check tests Check tests are similar to duplicate tests, but specimens which have been measured originally in an earlier batch are used. These tests are helpful to prove the stability and integrity of specimens taken from patients several hours, or even days, earlier. The interpretation is the same as in duplicate tests. The same specimens should be used for check tests and duplicate tests. If an SD calculated from the resulis of check tests is greater than for duplicate tests, this clearly indicates malfunction of instruments and/or reagents, provided that specimens are not altered. Replicate tests Replicate testing of a specimen evaluates technical excellence and/or stability of an instrument by measuring the reproducibility of a result. Like duplicate tests, replicate tests will37 not detect faults in pipetting devices and/or reagents. Precision may be obtained by calculating the coefficient of variation. Control chart A control chart is a simple diagrammatic presentation of results of an analyte measured using a control material of which the mean and standard deviation of measurements have been determined. A plot is made on arithmetical graph paper with the results of the analyte on the vertical scale against the date of measurement on the horizontal scale. The diagram gives an immediate overview of the quality of laboratory measurements. Accidental changes and systematic drifting in measurement are casily identified. They can be caused by improper function of the analytical system ‘¢ improper calibration of the analytical system ¢ inappropriate quality of the reagents © inappropriate quality of the control material ‘¢ inappropriate application of the analytical method analyte: month: 25D 28D I day 1 2.5 45.6 F 8 91011121314 181617 18 19.2021 22 2 2425 277287980 FF FIG.5: Control chart 1. Results are close to and equally distributed around the mean value indicating good laboratory performance. ILA result outside the 2 SD limits, indicating that the analytical system is out of order. Immediate action should be taken to identify the cause. Ii, Sequence of results within the limits of acceptance but uniformly distributed above (or below) the ‘mean, indicating a bias which needs to be identified and corrected. IV. A sequence of seven results with a tendency towards decreasing (or increasing) values, even when passing through the mean, indicating a steady tendency of the analytical system (= instrument performance, reagents, calibrators, control material) to depart from control. Causes need to be identified and corrected. V. Broad scattering of the results around the mean indicates low precision of measurement, the causes of which need to be identified‘The control chart should be prepared for each analyte measured in a laboratory (Fig. 5). The use of the control chan is mandatory for each laboratory that does not use more sophisticated techniques for internal quality control. The mean value of a material used for control of precision of measurement is marked at the centrai line. The control chart can also be used for control of accuracy, if the value given for an analyte in the control material is calibrated against a reference material with a true value. If a material is used for contrat of accuracy, the central line is marked with its assigned value. If two different materials are used for control of precision and control of accuracy, two different control charts for each material must be prepared. The upper and lower lines indicate the upper and lower limits of acceptance, usually twice the standard deviation (2 SD) above and below the mean, as determined in a preliminary investigation for precision from a series of repetitive measurements. The assigned value and limits of acceptance are provided with commercial control materials. They must be determined in locally produced control material. The results of each actual measurement of control material are plotted on a control chart. The pattern of plots can reflect different situations, some of which should result in immediate corrective action in the analytical system. The different situations are shown in Fig. 5. Patient data In cases where no significant day-to-day or week-to-week variability in the means of a specific analyte from a number of investigations in specimens from different patiems is observed, and the tests are not selectively biased, the result from an individual patient may be compared with previous results from the same patient, measured within a certain time interval in order to collect information about the quality of the most recent result in measurement (see also Chapter 3). Intralaboratory and interlaboratory comparison In an intralaboratory comparison two samples of a single specimen are analysed by different reagents, instruments or operators, or even by different analytical procedures. The analysis can be performed simultaneously or consecutively, at identical laboratory sites or elsewhere. The results are to be handled in the same manner as duplicate tests. Patients’ samples or a matrix containing an aqueous, control material are suitable for an intralaboratory comparison, The results from a series of duplicate or replicate measurements must be handled in the same way. With regard to interlaboratory comparison, a powerful instrument of internal quality assurarice is the exchange of samples or of a single specimen between laboratories, provided that the samples have not altered and comparable methodology is used.Control ef precision To ensure the precision of reagents, one single vial or test strip of each lot may be analysed. This also applies to taking 10 replicate tests for the first evaluation of each new instrument, and from a single lot for each specified analyte. These measurements may be repeated at suitable time intervals, depending on the technical requirements for the requested investigations, ‘The materials for the control of precision may be purchased or locally produced. Remaining samples of serum or plasma can be collected each day, pooled and frozen. After a certain period of time (1-12 weeks, depending on the amount of material and the stability of the analyte), all frozen samples are thawed, pooled, portioned in aliquot volumes (2 or 5 mL, depending on the daily volume needed) and frozen again. The concentration or activity of an analyte may be measured over S~15 consecutive days to determine precision in the performance of analysis. It may be sufficient to plot the results of measurements on the quality control chart every day and calculate the data retrospectively after a suitable period of time (e.g. one week). The contro! materials must maintain sufficient stability of the analytes to be measured. Their composition should be similar to those in the patients’ specimens. Control materials should not be used for calibration of a method and/or an instrument. The concentration or activity of a specified analyte may be chosen at the borderline of the reference range. For routine investigations, control of precision must be made in each series of measurements. The shortest series contains a single patient's sample. In longer scries of measurements, controt samples should be inserted after each segment of 10 to 20 samples from patients. The precision measurement of a specified analyte is acceptable if the relative standard deviation (= coefficient of variation) is smaller than the requirements in clinical decision-making. Every new batch of control material should be compared with the control material in usc prior to its introduction for the purpose of quality control. The mean and standard deviation should be calculated from the results of the control materials at timed intervals (15 results every four months ar the longest). In case of low precision the equipment, reagents and methodology as well as the calibration material need to be thoroughly reassessed. Control of accuracy The materials for control of accuracy can also be produced by the actual operator. Care has to be taken in evaluating the target value. The use of accuracy control materials produced by manufacturers with given assigned values, preferably reference method values, provides additional security for the operator. Accuracy control does not necessarily need to be assessed at every run. It may be made on each fourth or eighth series of measurements every day, week or month, depending on the stability of the analytical system, Accuracy must be checked when changing reagents,