Samples: from the Patient to the Laboratory

3rd, revised edition 2003

Samples: From the Patient to the Laboratory

The impact of preanalytical variables on the quality of laboratory results

W. G. Guder · S. Narayanan · H. Wisser · B. Zawta

Samples: From the Patient to the Laboratory. W. G. Guder, S. Narayanan, H. Wisser, and B. Zawata Copyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-30981-8 1st Edition, 1996 2nd Edition, 2001 3rd Edition, 2003

Front Cover: Fractal image from Mandelbrot’s non linear mathematics. Stephen Johnson; Tony Stone Bilderwelten, Munich

This book was carefully produced. Nevertheless, author and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

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Printed in the Federal Republic of Germany. Printed on acid-free paper.

Composition 4t Matthes + Traut Werbeagentur GmbH, Darmstadt Printing Druckhaus Darmstadt GmbH, Darmstadt Bookbinding Litges & Dopf, Buchbinderei GmbH, Heppenheim ISBN 3-527-30981-0 Preface and Acknowledgements

The authors having known each other for many years decided to summarize their ex- perience of preanalytical variables in 1992 after observing an increasing contribution of these factors on laboratory results. They agreed to summarize their knowledge in a short and understandable form aiming to increase the awareness of these factors among all professions involved in the preanalytical phase of the laboratory diagnostic process. This idea was generously supported by Becton Dickinson, Europe.

After the style and general contents of the book were agreed upon in a first meeting of the authors together with the publisher, the manuscripts were completed by the authors in a short time with the help of many collaborators and colleagues. The authors would especially like to thank Heidrun Dürr and Edith Rothermel, Heidel- berg, Klaus Krischok, Munich, Ulrich Wurster, Hannover for providing and design- ing figures. Thanks also to Ingrid Freina, Ulrike Arnold and Patrick Bernhard, Munich, Carol Pirello, New Jersey, Kerstin Geiger, Marion Wajda and Helga Kall- meyer, Mannheim, Annelies Frim, Stuttgart for their expert secretarial help. David J. Purnell, Plymouth, Wolfgang Heil, Wuppertal, and James Brawley, Gaiberg/Heidel- berg greatly supported our work by critically reading the manuscripts. We would like to thank Alois Jochum for translation support.

The present 3rd version includes a special edition of “The Quality of Diagnostic Sam- ples“ as CD-ROM, containing all Recommendations of the Working Group on Pre- analytical Quality, updated May 2003, kindly provided by Chronolab AG, Zug, Switzerland. Several Figures have been replaced by the newest versions available and references adapted to more recent publications.

In continuation of a 10 years collaboration with the Publisher GIT we thank A. Pill- mann (Wiley-VCH) for her experienced support in editing this new version in close collaboration with all contributors.

The authors do hope that the new version will help to continuously increase the awareness of preanalytical variables as a possible source of laboratory errors. As the previous editions it is devoted to all professions involved in the organization and performance of preanalytical steps. The authors would be pleased if this work helps to improve the quality of patient care by increasing knowledge on preanalytical variables in the laboratory diagnostic process.

Walter G. Guder Sheshadri Narayanan Hermann Wisser Bernd Zawta

May 2003

III Contents

Samples: From the Patient to the Laboratory The impact of preanalytical variables on the quality of laboratory results

Preface and Acknowledgements ...... III Contents ...... V Foreword to the First Edition ...... IX

Dream and reality – An introductory case ...... 02 The importance of the preanalytical phase ...... 05

Biological Influences

Something unavoidable – Influences of age, gender, race and pregnancy ...... 06 Changing habits – Influences that can vary (diet, starvation, exercise, altitude) ...... 08 May I take a coffee, smoke or drink before blood sampling – Stimulants and addictive drugs as biological influence factors ...... 12

Collection of Specimen

When to test? – Timing of sampling ...... 14 Sampling during infusion therapy? – The impact of the sequence of diagnostic and therapeutic procedures .... 16 Sampling in the supine or upright position? – Effects of posture and tourniquet ...... 18 What site for sampling blood? – Phlebotomy, arterial puncture and sampling from catheters ...... 20 Blood from the skin – Capillary sampling ...... 22 Did the lab mix up my sample? – Techniques of sample identification ...... 24 A precious sample – Cerebrospinal fluid (CSF) ...... 26 A sample that is nearly always available – Urine and saliva as diagnostic probes ...... 28 Plasma or serum? – Differences to be considered ...... 32 Take a lavender tube! – Additives and colour codes ...... 34

Transport and Storage

Fax me a sample – Effects of time and temperature during transport ...... 36

V Contents

Samples in transit – Legal standardization for mailing samples ...... 38 How to keep a sample “fresh“ – Storage of samples in the laboratory ...... 40

Preparation of Samples for Analysis

What’s has to be done on specimen arrival? – Specimen processing, centrifugation, distribution ...... 42 Continuous or batchwise? – Preanalytical workflow and robotics ...... 44 Safety aspects during the preanalytical phase – Disposal of specimens, needles, tubes and chemicals ...... 46

Special Aspects with each Analyte

What is needed before blood transfusion? – Special aspects in immunohaematology ...... 50 Why a special tube for coagulation tests? – Special aspects in haemostasiology ...... 52 Blood cells are sensitive! – Special aspects in haematological analysis ...... 54 Everything from a drop of blood? – Special aspects in clinical chemistry ...... 56 Special tubes for hormones? – Preanalytical factors in immunoassays ...... 58 Blood cells can provide important information – Special aspects in cellular analysis ...... 60 How to handle genes – Special aspects in molecular biology ...... 62 When gases evaporate – Special aspects for blood gases and ionized calcium ...... 66 The right time for drugs… – Special aspects in therapeutic drug monitoring (TDM) ...... 68 Bacteria and viruses – Special aspects in microbiology ...... 72

VI Contents

Endogenous and Exogenous Interferences

Can turbid samples be used? – Effects of lipemia ...... 76 A difficult case – Pitfalls with endogenous antibodies ...... 78 The serum sample looks reddish – Effects of haemolysis ...... 80 Does the laboratory have to know all my drugs? – Mechanisms and treatment of drug interference ...... 82 Everything under control? – Quality assurance in the preanalytical phase ...... 84

References ...... 88 Glossary ...... 97 Index ...... 102

The Quality of Diagnostic Samples CD-Rom Annex

Serum, Plasma or Whole Blood? Which Anticoagulants to Use? The optimal sample volume Analyte stability in sample matrix The haemolytic, icteric and lipemic sample Samples and stability of analytes in blood, urine and CSF

Genie, Microtainer, Pronto and Safety-Lok are trademarks of Becton Dickinson and Company

VII Foreword to the First Edition

aboratory tests generally provide a more sensitive indicator of the state of a patient‘s health than the patient‘s account of how he or she feels. This has L prompted an increasing emphasis on laboratory tests in the diagnosis and management of the patient‘s disease. Major decisions about the management of a patient are being made on small changes in laboratory data. Thus, a decision to change the dose of a patient‘s drug is often made on its plasma concentration. Laboratories have long been aware that many non-disease factors may affect clinical laboratory test values. These include the potential effect of drugs, either through an effect on the physiological function of various organs, or an interference with an analytical method. Whereas the laboratorian may be aware of the possibility of an analytical interference, clinicians are largely unaware of these effects and the available resources to help them interpret test values correctly. When this information is not given with the result, clinicians may misinterpret test values and take an inappropriate action with their patients. Clinical decisions based on laboratory test values are correctly made only when the conditions under which blood or other specimens are properly identified and standardized, or when the lack of standardization is recognized and allowances are made for some lack of comparability with previous test values. While laboratorians are aware of the concepts of intra- and interindividual variation as they affect laboratory data, many colleagues are unfamiliar with all but the most obvious causes of differences in test values, such as gender and age. An understanding of intraindividual variation of test values is important if appropriate clinical decisions are to be made when serial data are being followed. The new concepts of critical differences or reference changes are now important. For proper interpretation of the typically small differences between laboratory data obtained on successive specimens from patients, the variables affecting the test values need to be standardized wherever possible, but first the pertinent variables need to be identified. These are the issues that prompt the need to revisit all the factors related to preanalytical variables. It is thus particularly timely for this book to be published. The authors hope to reach a broader audience than the laboratorians who are probably quite familiar with many of the factors affecting test results. Since 1956, when Roger Williams published his pioneering studies on the differences between people in a book entitled “Biochemical Individuality“, physiologists have been concerned with the differences between people. Now that we have a broader understanding of the genetic influence on human physiology and behavior and a greater need to extract more information from small changes in laboratory data, the publication of a new book concerned with preanaytical variables which contribute to intra- and interindividual variability is both timely and welcome. This book is intended not just for laboratorians but also for physicians, nurses and everyone involved in the chain of events from the decision to order a laboratory test to the interpretation of its results. Proper application of the information contained in this book should lead to less un- necessary testing, reduced costs and a better understanding of the results.

Philadelphia, April 1996 Donald S. Young M.D., Ph.D.

IX Dream and reality

A new patient with diabetes Two blood samples are drawn from the mellitus is encountered patient the following morning (after she has fasted for 12 hours), from the ante- Mrs. Haseltine is a 56-year-old lady cubital vein into closed tubes, one, with who lives in a remote area. She consults a lavender-colored stopper, containing her nearby practitioner and reports that EDTA, the other, with a green cap, over the last two weeks she has urinat- containing heparin. Mrs. Haseltine is ed more frequently than usual. Also, informed that she has type II diabetes her body weight has decreased, al- mellitus and will have to be placed on though she “drinks more soft drinks than a diet in order to treat her disease. She ever before“. The practitioner finds a is asked to phone the next day to positive dipstick result for glucose in obtain information on her laboratory her urine. Using a glucometer, he results and for further advice. measures glucose from fingertip blood obtained by pricking with a fine lancet. In the meantime, the heparin blood The first drop of blood is washed away sample has been centrifuged to sepa- with a swab of gauze. In the following rate plasma from the cellular elements. drop, glucose is measured by the meter, Both tubes are sent to the laboratory by a process that takes about 30 seconds. courier in a container especially de- The result is 280 mg/dL (15.56 mmol/L), signed to keep samples at constant tem- far above the upper limit of the normal perature. The laboratory receives the range. Mrs. Haseltine is informed that samples together with the patient’s data she may have diabetes mellitus and is and requests for determinations: glycat- referred to a diabetologist the next day. ed haemoglobin and blood cell counts from the EDTA blood; potassium and creatinine from the plasma, which has The right sample for the right test been separated from blood cells, in the at the right time closed heparin tube.

The diabetologist confirms the result The laboratory technician identifies all obtained by the practitioner using a the samples by comparing the name capillary blood sample taken 1 hour and bar code number with those on the after breakfast. request sheet. He then enters the request Fig. 1-1 into the lab computer. The samples are put into bar code-reading analyzers for identification and performance of the requested tests. A subsample is taken from the EDTA blood – after slowly mixing it for 3 min on a roller mixer – for the determination of haemoglobin

A1c by chromatography. The laboratory report, shown in Tab. 1-1 , is sent to the diabetologist the next morning.

02 Samples: From the Patient to the Laboratory. W. G. Guder, S. Narayanan, H. Wisser, and B. Zawata Copyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-30981-8 An introductory case

Tab. 1-1 Laboratory report late, however, and misses her morning Haseltine, Elsa July 13, 1994 10 a.m. appointment. She arrives at the doctor’s Patient Reference office at noon, having had a snack on Result Interval the way. She is stressed when the nurse offers her a glucose-containing drink Haemoglobin A 8.5% 2.5–6.0 1C after taking a “fasting“ blood specimen. Haemoglobin 14.4 g/dL 12–15 Potassium 3.6 mmol/L 3.5–4.5 She feels nauseated while slowly consum- Creatinine 1.0 mg/dL Below 1.1 ing the drink. Whilst waiting for the nurse, she decides not to drink it all and empties Mrs. Haseltine is taken into the neigh- the remaining drink down the bathroom boring county hospital together with a sink. Of course, she doesn’t report this letter from the diabetologist informing incident to the nurse when she returns to the clinician about all tests performed take a capillary blood sample at one and and the results obtained. After two two hours after the first sample. weeks of treatment, Mrs. Haseltine has learned to control her blood sugar us- When the results are shown to the doctor ing a small glucometer. No further (Tab. 1-2 ), he realizes that the glucose treatment was needed for the next few concentrations after the first and se- years. cond hour are not that much different. The diabetologist, unable to arrive at a diagnosis, asks the patient to report the Fig. 1-2 following day at which time two venous blood samples are collected, one with a lavender-colored stopper and the other with a green cap. The tubes are sent to a private laboratory by car. Next day, the results shown in Tab. 1-3 are received by telefax together with the reference values for each test. The glucose value is now normal, potassium el-

evated and haemoglobin A1c, an indicator of mean blood glucose, elevated to diabetic levels. The diabetologist, This is what might happen in reality concerned by the high potassium level, refers the patient to a clinic. This institu- Mrs. Haseltine goes to the practitioner tion diagnoses that the patient has type with the same symptoms for the same II diabetes mellitus, based on their la- condition. In contrast to the positive urine boratory results. dipstick result for glucose, the blood sugar is nearly normal (120 mg/dL). Tab. 1- 2 The practitioner, to play-it-safe, again Results in doctors office: glucose tolerance test refers the patient to a diabetologist. Haseltine, Elsa July 12, 1994 – 2 p.m. Fasting Glucose 160 mg/dL One week later, Mrs. Haseltine is called 1-Hour Glucose 110 mg/dL in for a glucose tolerance test. The only 2-Hour Glucose 120 mg/dL advice she is given is to fast the night before the test. Mrs. Haseltine wakes up

03 Dream and reality

Fig. 1-3 She did not report this to the doctor or the nurse, because she wasn’t aware of the possible influence of this snack. For the same reason, she did not report not consuming all of the glucose drink, which had led to a decrease rather than an increase of blood glucose after one hour. The “increase“ at the second hour may have been due either to method variation or to a reactive increase brought about by metabolic reactions in the late afternoon. Normally, a glucose tolerance test is performed in the morning, the reference values be- Tab. 1-3 Report from private laboratory ing valid only for the morning. It should Haseltine, Elsa – July 13,1994 3 p.m. be carried out under standard condi- Units Patient's Result Reference Range tions, as recommended by national and international expert panels. HbA1c % 8.5 3.5 – 6.0 Haemoglobin g/dL 13.5 12 – 15 Potassium mmol/L 5.8 3.5 – 4.5 Why was potassium elevated and Creatinine mg/dL 1.0 Below 1.1 glucose normal in the venous Glucose mg/dL 105 70 – 110 specimen?

What happened to Answer: The sample was transported in Mrs. Haseltine's samples? contact with the cells for over two hours in a non- air conditioned car on a hot Undoubtedly, Mrs. Haseltine was in a day. This caused the blood cells to diabetic state. Why was the fasting metabolize glucose and release potas- blood sugar nearly normal? sium, the concentration of which is approximately 40 times higher in cells Answer: Fasting may result in near than in plasma. This in-vitro influence normal values in type II diabetics. In makes unstabilized blood unsuitable this case, the nurse took the first drop for glucose determination. Potassium can of blood from a fingerprick after be reliably measured only if plasma is “milking“ the finger to obtain sufficient promptly separated from the cells. blood. All these errors could have been prevented had the preanalytical phase Why was the result of the glucose been strictly controlled. Mrs. Haseltine tolerance test inconclusive? would have been diagnosed earlier with less stress, and fewer costs would Answer: The first result was related to have been incurred. patient stress, which leads to increased amounts of glucose being released from liver glycogen stores. Moreover, Mrs. Haseltine had a snack on her way to the doctor because she was hungry.

04 The importance of the preanalytical phase

This book is intended to increase coagulant, the optimal awareness of the importance of all sample volume and steps of the preanalytical phase, in- the stability of an- cluding patient preparation, sampling, alytes in sample transport and storage of patient samples. matrix is included in the Annex: The In each chapter, covering two opposite Quality of Diagnos- pages, possible preanalytical variables tic Samples. are explained with regard to mechanisms, effects and preventive actions intended to prevent misinterpretation of laboratory results. In the respective chapters, warnings are given in red and recommendations in green. Like disease mechanisms, biological influences can change the concentration of measured analytes in-vivo, whereas in-vitro changes have to be separated into changes undergone by the measured analyte and interference of the method used to measure the analyte. These definitions are important, because only the latter can be avoided by using a more specific method. The interested reader is referred to the literature summarized on pages 88–95as well as the glossary which defines all the special terms used in this book (p. 97). A detailed information on preanalytical variables of all analytes together with the recommendations on the choice of anti-

Optimal treatment of patient and his samples is defined as the gold standard

05 Something unavoidable

Intrinsic influences such as race, gender phatase activity (AP) in serum (which and age may influence target analyte peaks during the growth phase, mirror- concentrations in clinical chemistry and ing bone osteoblast activity) and total haematology. These variables are indi- and LDL-cholesterol. In addition, age- vidual features of a subject and hence dependent AP activity and LDL- and not subject to change. Quite often, HDL-cholesterol in serum are influenced intrinsic and external factors are difficult by gender. These gender differences in to distinguish. turn change as a function of age.

Fig. 2-2 Age creatine kinase α-amylase granulocytes Influence of race on U/L U/L G/L creatine kinase, Age may affect blood and urine ana- amylase and lyte concentrations after birth, during 300 granulocytes in blood. adolescence or in old age (Fig. 2-1). 200 4 P = pancreatic Erythrocyte counts and hence haemo- 200 isoenzyme globin are much higher in neonates S = salivary compared to adults. Within the first few + 3

isoenzyme + + days following birth, increased arterial + ▼ PS PS oxygen provokes erythrocyte degrada- PS tion. The resulting increase in haemoglobin leads in turn to enhanced concentrations of bilirubin. Since liver white west- indian asian hispanic asian black british black white function (here in particular glucuronidation) is not fully established in neonates, increased concentrations of Race Fig. 2-1 bilirubin are observed. Fig. 2-2 illustrates examples of analytes Age dependence of which are affected by race. Black Ameri- various substrates and Uric acid concentrations in neonates are cans of both genders have significantly enzyme activity (8, 35). in a range similar to adults. However, lower white blood cell counts compared Alkaline phosphatase within days after birth, a significant de- to whites. This difference is readily was measured at crease is observed. Other examples of explained by a reduction in the number 30 °C (86 °F) ▼ age-dependence include alkaline phos- of granulocytes. In contrast, haemoglobin, haematocrit and lymphocyte counts are almost identical in both g/L U/L mmol/L alkaline groups (97). The monocyte count in 200 haemoglobin phosphatase 8 mol/L 800 whites exceeds that of blacks (11). A µ 300 7 significant difference in creatine kinase uric acid (CK) activity has been observed for 600 6 200 + both genders in black and white peo- 5 ple. This difference is not due to differ- 100 160 cholesterol 400 4 ences in age, height or body weight 140 + (81). A substantial difference in amy- 3 100 LDL-cholesterol lase activity has been established be- bilirubin 200 2 tween West-Indians and native Britons. 60 + 1 Based on the generally accepted 20 + HDL-cholesterol threshold value, 50 percent of West In- 246 6 810121416 18 15 25 35 45 55 dians had elevated amylase activities birth days years years (219). Significant racial differences have

06 Samples: From the Patient to the Laboratory. W. G. Guder, S. Narayanan, H. Wisser, and B. Zawata Copyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-30981-8 Influences of age, gender, race and pregnancy been reported for serum concentrations triglycerides of vitamin B12 (1.35 times higher con- creatine kinase γ-glutamyltransferase centrations in black people) (183) and bilirubin Lp(a) (2 times higher concentrations in alanine aminotransferase creatinine blacks compared to whites). It is note- myoglobin uric acid worthy in this context that neither ather- urea ammonia osclerosis nor mortality is higher in aspartate aminotransferase haemoglobin blacks with high Lp(a) (75). acid phosphatase erythrocytes amino acids alkaline phosphatase Gender cholinesterase iron glucose As with many macroscopic features LDL-cholesterol albumin and gender-specific hormone patterns, immunoglobulin G cholesterol gender differences can likewise be total protein reticulocytes found in clinical chemistry and haema- apolipoprotein AI copper tology (Fig. 2-3). The gender difference prolactin of serum iron concentrations disappears HDL-cholesterol in patients older than 65 years. Other 0.80.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 examples of gender differences are CK and creatinine. The serum activity or Fig. 2-3 concentration depends on muscle mass changes in hormone production and Male – female which is in general more pronounced in the plasma concentrations of fertility differences related to males. Certain athletic activities which hormones during pregnancy are ac- the mean value of lead to increased muscle mass may companied by changes in various ana- females as given in (35) blunt this difference (see p. 9–10). lytes, e.g. thyroid hormones, metabolites (amino acids↑, urea↓), electrolytes Pregnancy (calcium↓, magnesium↓, iron↓, zinc↓, copper↑), proteins (especially acute When interpreting laboratory results phase proteins↑), and some diagnosti- during pregnancy, it is necessary to cally important lipids (triglycerides↑, take into account the gestational week cholesterol↑), enzymes (alkaline phos- at which each sample was taken. phatase↑, cholinesterase↑), factors of the plasma coagulation system and compo- During a healthy pregnancy, the mean nents of the fibrinolytic system. The se- plasma volume rises from about 2.6 L to dimentation rate is increased five-fold 3.9 L, with probably little change occur- during pregnancy. The concentration ring in the first 10 weeks of gestation, changes are caused by different mech- and a subsequent progressive rise up anisms as increased synthesis of trans- to the 35th week, at which time the val- port proteins, increased metabolic turn- ues level off. Tab. 2-1 describes the over rate or dilution. mechanism underlying the changes in plasma during pregnancy.

The urine volume may also increase physiologically by up to 25% in the 3rd trimester. There is a 50% physiological increase in the glomerular filtration rate in the last trimester. The well-known

07 Changing habits

Diet depends on the composition of the Fig. 3-1 food and the elapsed time between Change of the serum concentration of dif- Diet and drinking are major factors sampling and food intake. The serum ferent analytes two influencing a number of analytes in concentration of cholesterol and triglyc- hours after a standard clinical chemistry. From a practical erides are influenced by various factors meal (37, 206) point of view, one should distinguish as food composition, physical activity, ▼ acute effects from those observed over smoking, consumption of alcohol and coffee (51). Elevated levels of ammo- triglycerides 1.78 aspartate aminotransferase 1.25 nia, urea and uric acid are observed bilirubin 1.16 during a high protein and nucleotide glucose 1.15 phospate 1.15 diet. The changes occurring after a alanine aminotransferase 1.055 potassium 1.052 standard carbohydrate meal (75 g) are uric acid 1.027 diagnostically helpful in testing glucose total protein 1.018 albumin 1.018 tolerance. On the other hand malnutri- calcium 1.016 sodium 1.004 tion and starvation may alter analyte alkaline phosphatase 1.004 concentrations in a clinically relevant cholesterol 1.000 urea 1.000 fashion. Early indicators of low protein lactate dehydrogenase 1.000 diet are reduced serum concentrations 1.0 1.25 1.5 1.75 2.0 of prealbumin and retinol-binding pro- Relative change in concentration after/befor meal tein. Some alterations in clinical chemi- a longer period. A critical question in cal analytes induced by starvation over daily routine is whether a standard meal 48 hours are summarized in Fig. 3-2. affects target analytes. Fig. 3-1 shows Metabolic acidosis with a decrease of the percentage change in different both pH and bicarbonate results from analyte concentrations as a function of an increase in organic acids, mainly Fig. 3-2 food intake (37, 206). Effects of 5 per- the ketone bodies (acetoacetic acid, Variation of several cent and less may be neglected, since 3-hydroxybutyric acid). analytes after 40-48 h starvation (113). they are clinically irrelevant. Therefore, * Starting point after samples for these analytes do not re- Starvation 14 h starvation quire strict food deprivation. The extent ▼ of food-induced alterations in analytes Changes in analyte concentrations induced by long-term starvation (4 weeks) 3-hydroxybutyrate* are shown in Fig. 3-3 at the end of the 30x starvation period in comparison to the initial values. The concentrations of blood cholesterol, triglycerides and urea are reduced. In contrast, creatinine and uric 10x acid concentrations are elevated. The increase in uric acid concentration dur- acetoacetate* ing starvation periods even requires treatment. The latter is due to reduced Increase (x-fold) clearance of uric acid as a result of ke- 5x tonemia (44). It is readily apparent that free fatty acids long-term starvation is closely associ- pyruvate*, lactate* ated with reduced energy expenditure; glycerol hence, as a result, T4 and, to a larger glucagon 1x extent, T concentrations are reduced in insulin 3 14 h 48 h serum. Besides such alterations, urinary

08 Influences that can vary (diet, starvation, exercise, altitude) excretion of several compounds is like- deviation (%) wise affected by long-term starvation. –60 –40 –20 0 20 40 60 Urinary excretion of ammonia and cre- sodium atinine is increased whereas that of potassium urea, calcium and phosphate is reduced calcium chloride (231). Changes in analyte concentra- protein albumin tions brought about by long-term starva- glucose tion are similar to those observed uric acid urea

following surgical procedures or in analytes creatinine cholesterol patients with a catabolic status. triglycerides alkaline phosphatase alanine aminotransferase In measuring quantitative urinary excre- aspartate aminotransferase γ-glutamyltransferase tion rates, excreted amounts per day haemoglobin haematocrit are preferable to those per liter in order weight loss to eliminate variations in drinking habits and water excretion. longer duration (e.g. running, swimming, Fig. 3-3 cycling) which utilizes ATP produced by Change (%) of clinical aerobic or anaerobic pathways. In ad- chemical analytes after Mechanisms dition, the effect of physical training 4 weeks starvation Changes may be due either to an and muscle mass should be mentioned. and a daily supply of increase in reabsorption of the measured Acute changes of analytes during exer- 33 g protein, vitamines analyte (triglycerides, glucose, amino cise are due to volume shifts between and electrolytes (44, 232) acids), intestinal or liver metabolism of the intravasal and interstitial compart- reabsorbed metabolites (VLDL, urea, ments, volume loss by sweating and ammonia) or regulatory changes due changes in hormone concentrations to food intake or deprivation (uric acid, (e.g. increase in the concentrations of γ-glutamyltransferase, cholinesterase, ephinephrine, norepinephrine, glucagon, thyroxine, retinol-binding protein, ketone somatotropin, cortisol, ACTH and de- Fig. 3-4 bodies). creased concentrations of insulin) (4, Increase of various analyte concentrations 177). These changes in hormone levels after a marathon race. may in turn alter the leukocyte count to Recommendation Blood was drawn one more than 25 G/L as well as increasing day before and 45 min In order to avoid misinterpretation of glucose concentrations. Fig. 3-4 shows after the race (203) laboratory results, sampling after 12 h fasting and reduced activity is recommended as a standard procedure. 1x 2x 3x 4x potassium sodium Exercise alkaline phosphatase calcium Before considering the influence of ex- albumin ercise on target analytes in clinical glucose chemistry, two types of exercise have to inorg. phosphate be distinguished. First, static or isomet- uric acid ric exercise of brief duration and high urea intensity which utilizes the energy (ATP aspartate aminotransferase and creatine phosphate) already stored pyruvate kinase in muscle and, second, dynamic or iso- creatine kinase tonic exercise of lower intensity and

09 Changing habits

changes in analyte concentrations creased capacity of the oxidative en- induced by marathon running (203). The zyme system. This effect in turn increases extent of change depends on a variety the capacity of the muscle to metabo- of individual and/or environmental fac- lize glucose, fatty acids and ketone tors (e.g. training status, air tempera- bodies in aerobic pathways. As a conse- ture and intake of electrolyte- and car- quence, mitochondrial CK-MB increases bohydrate-containing liquids during the to more than 8 percent of the total CK actual run). activity without evidence of altered myocardial function. Well-trained individu- The changes observed (e.g. increased als have a higher percentage of total albumin) can in part be attributed to activity in terms of the CK-MB of skeletal the above-mentioned volume shift from muscle compared to untrained persons. intravasal to the interstitium or to loss of Several other analyte concentrations volume by sweating. The increased uric likewise depend on muscle mass and acid concentration in serum is a conse- training status. Thus, plasma creatinine, quence of reduced urinary excretion urinary creatinine and creatine excre- due to increased lactate concentrations. tion increase and lactate formation after Hypoxia-mediated creatine kinase (CK) exercise decreases in trained com- increase depends on the training status pared to untrained athletes. Vigorous and hence shows a high degree of indi- exercise may cause erythrocytes or oth- vidual variability. The less physically fit er blood cells to be excreted in urine. an individual is the more pronounced These exercise-induced changes, how- the increase in CK. Training increases ever, usually disappear within a few both the number and the size of mito- days. chondria which is associated with in-

10 Influences that can vary (diet, starvation, exercise, altitude)

Altitude acid. Adaptation to altitude takes Some blood constituents exhibit signi- weeks and return to sea level values ficant changes at high altitude com- takes days. A significant decrease in pared to findings at sea level. values with increasing altitude is found in the case of urinary creatinine, creati- Significant increases with altitude are nine clearance, estriol (up to 50% at observed, for example, for C-reactive 4200 m) serum osmolality, plasma protein (CRP) (up to 65% at 3600 m), renin and serum transferrin (239). β2-globulin in serum (up to 43% at 5400 m), haematocrit and haemoglobin (up to 8% at 1400 m) and uric

11 May I take a coffee, smoke or drink before blood sampling?

Caffeine account for concentration changes by Caffeine is found in many constituents direct or indirect effects. Decreased an- of food ingested daily. Despite its wide- giotensin converting enzyme activity spread use, the influence of caffeine on (ACE) in smokers is believed to result various analytes in clinical chemistry from the destruction of lung endothelial has not been investigated in detail. cells with a subsequent reduction in the Caffeine inhibits phosphodiesterase and Fig. 4-1 -40 -30 -20 -10 0 +10 +20+30+40+50+60 (%) hence cyclic AMP degradation. Cyclic Deviation (%) of blood AMP in turn promotes glycogenolysis, angiotensin converting enzyme analyte concentrations prolactin between current thereby increasing blood glucose con- β-carotinoids pyridoxal phosphate smokers and non centrations. In addition, the glucose selenium smokers, chronic concentration increases due to gluco- HDL-cholesterol LDL-cholesterol effects (239) neogenesis via epinephrine. Activation cholesterol haematocrit ▼ of triglyceride lipase leads to a three- MCV fibrinogen fold increase of non-esterified fatty copper red cell mass acids (141). Quantification of hormones cadmium lead and drugs bound to albumin is hamper- monocytes lymphocytes ed by the fatty acid-induced displace- neutrophils ment effect. Three hours after the intake CEA of 250 mg of caffeine, plasma renin release of ACE into the pulmonary cir- activity and catecholamine concentra- culation and/or enzyme inhibition (76). tions have been found to be elevated The extent of changes also depends on (175). the amount, kind (cigarettes, cigars, Studies intended to investigate these pipes) and technique of smoking (with analytes should take caffeine consump- or without inhalation). Moreover, smok- tion into account. ing-induced changes are influenced by age and gender (204). Fig. 4-2 shows the concentrations of Effects of smoking cotinine, thiocyanate and carboxy- Smoking leads to a number of acute haemoglobin, used as markers for the and chronic changes in analyte con- qualitative and quantitative assessment centrations, the chronic changes being of smoking habits. Cotinine has the rather modest. Smoking increases the advantage of having a longer half-life plasma/serum concentrations of fatty (20– 28 h) than nicotine, the parent acids, epinephrine, free glycerol, al- compound (12– 15 min) (189). Fig. 4-2 dosterone and cortisol (239). These Effect of smoking on CO-haemoglobin concentration % changes occur within one hour of smok- different blood ana- 4 8 ing 1-5 cigarettes. Alterations in analytes cotinine concentration µg/L lytes caused by smoke constituents (141, 239) induced by chronic smoking include 200 400 blood leukocyte count, lipoproteins, the

▼ non CO-Hb activities of some enzymes, hormones, vit- low amins, tumor markers and heavy metals moderate (Fig. 4-1) (239). heavy cotinine The mechanism underlying these changes

has not been fully elucidated. A large thiocyanate number of pyridine compounds, hydrogen cyanide and thiocyanate are 100 200 found in tobacco smoke. They can thiocyanate concentration µmol/L

12 Stimulants and addictive drugs as biological influence factors

Alcohol -50 % changes +100 +200 Alcohol consumption, depending on its osteocalcin duration and extent, may affect a number prolactin ADH of analytes. These alterations are used cortisol acute effects ANP in part for diagnosis and therapeutic cholesterol monitoring. Among alcohol-related triglycerides aldosterone changes, acute and chronic effects LDL-cholesterol should be considered separately. VMA MCV chronic effects The acute effects (within 2–4 hours) of cholesterol triglycerides ethanol consumption are decreased cortisol serum glucose and increased plasma alanine aminotransferase estradiol lactate due to the inhibition of hepatic epinephrine gluconeogenesis. Ethanol is metabo- norepinephrine aspartate aminotransferase +260 lized to acetaldehyde and then to ac- γ-glutamyltransferase +1000% etate. This increases the formation of hepatic uric acid formation (67). plasma triglyceride breakdown. The Fig. 4-3 Together with lactate, acetate decreas- increased MCV may be related to a Acute and toxic effects es serum bicarbonate, resulting in direct toxic effect on the erythropoetic of alcohol ingestion on metabolic acidosis. Elevated lactate re- cells or a deficiency of folate (173). clinical chemical duces urinary uric acid excretion. Con- The data in Fig. 4-3 do not take into analytes (115, 168, sequently, after acute alcohol ingestion, account either the dose or the time- 239) the serum concentration of uric acid dependency, which underly both the acute increases (204). and the chronic effects. Enhanced diuresis The long-term effects of ethanol ingestion is also a result of the decreased release of include an increase in the serum activity vasopressin followed by increased secre- of liver enzymes. The increase of tion of renin and aldosterone (17, 115). g-glutamyltransferase activity is caused by enzyme induction. Glutamate dehydrogenase as well as aminotransferas- Addictive drugs es (AST, ALT) activities increase due to a Addictive drugs such as amphetamine, direct liver toxic effect (57). The increase morphine, heroin, cannabis and cocaine in desialylated forms of proteins in can influence the results of laboratory blood (i.e. carbohydrate deficient trans- tests. Morphine causes spasms of the ferrins) is due to an inhibition of enzy- sphincter of oddi, thus elevating levels matic glycosylation during post-transla- of enzymes such as amylase and lipase. tional processing of these proteins in The biological effects of addictive the liver. In chronic alcoholism, serum drugs on selected laboratory tests are triglycerides increase due to decreased listed in Tab. 4- 1

Tab. 4-1 Biological effects of addictive drugs on plasma concentrations of selected analytes (241) Addictive drug Increased/Decreased in plasma

1. Amphetamine Increased: free fatty acids. 3. Heroin Increased: pCO2, T4, cholesterol, 2. Morphine Increased: α-amylase, lipase, AST, potassium due to severe rhabdomyolysis.

ALT, bilirubin, alkaline phosphatase, Decreased: pO2, albumin. gastrin,TSH,and prolactin. 4. Cannabis Increased: sodium, potassium,urea, Decreased: insulin, norepinephrine, insulin, chloride. neurotensin, pancreatic polypeptide. Decreased: creatinine, glucose, uric acid.

13 When to test?

Changes brought about in specimens during the day and decreases at night due to the time factor should be taken (Fig. 5-2). into account in the preanalytical phase. Three questions are essential in this The cortisol rhythm may well be respon- context: sible for the poor results obtained from oral glucose tolerance testing in the ● When should a sample be taken? afternoon. – Time of day – Time after last sample Fig. 5-2 – Time after last meal µg/dL Daily variation of – Time after drug etc. 300 cortisol plasma concentrations of cortisol (shaded ● When do I require the result of the 250 areas = sleep period) specimen taken now? 200

● Can results be compared with the 150 results obtained at a different time in daily, monthly and yearly rhythms, ei- 100 ther from the same patient or from a reference population? 50

0 For the sake of clarity, we can differen- 06121824 h tiate between linear time, going from the past to the future, and cyclic time; For this reason, reference intervals are both of these can influence the results actually obtained between 7 and 9 of laboratory tests (Fig. 5-1). a.m. The circadian rhythm can also be influenced by individual rhythms con- Influence of circadian rhythm (217) cerning meals, exercise and sleep. These influences should not be confused with Several analytes tend to fluctuate in real circadian changes. In some cases, terms of their plasma concentration seasonal influences also have to be 1 over the course of a day (Tab. 5- ). considered. Thus, triiodothyronine (T3)is 20% lower in summer than in winter Thus, the concentration of potassium is (82) whereas 25-OH-cholecalciferol ex- lower in the afternoon than in the mor- hibits higher serum concentrations in ning, whereas that of cortisol increases summer (162).

Fig. 5-1 Linear and cyclic Analytes may change during the Chronobiological influence chronobiological menstrual cycle (239) influence Analytes can also exhibit statistically Linear significant changes due to the biological Cyclic (e.g. age) changes that occur in the hormone pattern during menstruation. Thus, the aldosterone concentration in plasma is Daily Biological (e.g. Seasonal (circadian) menstrual cycle) twice as high before ovulation than in the follicular phase. Likewise, renin can

14 Samples: From the Patient to the Laboratory. W. G. Guder, S. Narayanan, H. Wisser, and B. Zawata Copyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-30981-8 Timing of sampling

Tab. 5-1 Diurnal variation of selected analytes (S = serum; U = urine) (235)

Analytes Maximum Minimum Amplitude Analytes Maximum Minimum Amplitude (time of day) (time of day) (percentage (time of day) (time of day) (percentage of of daily mean) daily mean) ACTH 6–10 0–4 150–200 Norepinephrine (S,U) 9–12 2–5 50–120 Cortisol (S,U) 5–8 21–3 180–200 Haemoglobin 6–18 22–24 8–15 Testosterone 2–4 20–24 30–50 Eosinophils 4–6 18–20 30–40 TSH 20–2 7–13 5–15 Iron (S) 14–18 2–4 50–70

T4 8–12 23–3 10–20 Potassium (S) 14–16 23–1 5–10 Somatotropin 21–23* 1–21 300–400 Phosphate (S) 2–4 8–12 30–40 Prolactin 5–7 10–12 80–100 Sodium (U) 4–6 12–16 60–80 Aldosterone 2–4 12–14 60–80 Phosphate (U) 18–24 4–8 60–80 Renin 0–6 10–12 120–140 Volume (U) 2–6 12–16 60–80 Epinephrine (S) 9–12 2–5 30–50 Body temp. 18–20 5–7 0.8–1.0 °C

* Start of sleeping phase show a pre-ovulatory increase. Even interfering diagnostic procedures. Like- cholesterol exhibits a significant decre- wise, interfering drugs should be admin- ase during ovulation. In contrast, phos- istered after taking a blood sample. On phate and iron decrease during men- the other hand, in drug monitoring (see struation. p. 68) the exact timing of sampling is essential for correct interpretation of the drug level. Why has blood to be taken 12 hours after the last meal? (49, 239) Important rules for the timing of sam- As mentioned under diet (see p. 8–9), pling: several metabolic products of food can increase in venous blood or become ● If possible, samples should be taken altered due to post-absorptive hormonal between 7 and 9 a.m. effects. Other analytes may be disturbed ● Sampling should be carried out 12 by turbidity due to chylomicronemia hours after the last meal. present in postprandial blood samples. ● Samples should be taken before interfering diagnostic and therapeutic In order to eliminate these variables, a procedures are performed. 12 hour period of starvation is recom- ● In drug monitoring, consider the peak mended before blood is sampled for the after drug administration and the steady analysis of these analytes (Tab. 5-1 ). state phase before the next dose. ● Always document the exact time of sampling in the charts and requests. Timing with regard to diagnostic and therapeutic processes But: As described in the next chapter, a number of diagnostic procedures may ● A sample taken at the wrong time influence laboratory results. In order to can be worse than taking no sample. prevent this, the timing of sampling has ● A sample whose analytical results to be organized to take place before arrive too late is a wasted sample.

15 Sampling during infusion therapy?

Implausible laboratory results after Operations diagnostic and therapeutic inter- Changes in serum enzyme activities vention? are frequently so great that specific The following diagnostic and therapeutic targeting of an organ is no longer measures can result in both in-vivo possible. The elevation in acute phase (frequent) and in-vitro (less common) proteins (e.g. C-reactive protein (CRP), effects on laboratory tests (79, 99, 232): fibrinogen) at the beginning of the postoperative phase is accompanied ● Operations by a decrease in albumin; this cannot ● Infusions and transfusions be explained alone by haemodilution. ● Punctures, injections, biopsies, palpations, whole-body massage Transient elevations in urea concentra- ● Endoscopy tion in serum/plasma (up to 60 mg/dL ● Dialysis or 10 mmol/L) as well as a decrease in ● Physical stress (e.g. ergometry, cholesterol are very frequent in the first exercise, ECG) postoperative days whilst the creatinine ● Function tests (e.g. oral glucose concentration remains normal. This may tolerance test) be due to protein breakdown subsequent ● Immunoscintigraphy to gastro-intestinal tract surgery as well ● Contrast media, drugs as to bleeding in the lumen of the ● Mental stress bowel, e.g. in the case of a stress ulcer. ● Ionizing radiation

Tab. 6-1 Infusions/transfusions as interfering factors and/or contaminants of laboratory diagnostic tests

Infusion/Transfusion Analyte affected Trend Comments, Mechanism Dextran Thrombin time, reptilase time ↓ 5 – 10 sec slower von Willebrand factor ↓ Total protein in serum, plasma ↑ Biuret, method-dependent (turbidity, flocculation, greenish coloration) Urea, serum ↓ Blood grouping serology Pseudoagglutination γ-globulin Serological determinations during False positive virus-mediated and bacterial infections Electrolytes Potassium, sodium, magnesium ↑ Contamination Glucose Glucose ↑ Contamination Glucose Inorg. phosphate, potassium, ↓ Insulin Amylase, bilirubin ↓ Up to 15 %, particularly in neonates Fructose Uric acid ↑ Metabolic effect Citrate (blood transfusion!) pH value in blood ↓ Coagulation tests ↓↑ Inhibition

16 Sequence of diagnostic and therapeutic procedures

Infusions, transfusions Sampling for coagulation tests from heparin-contaminated catheters is par- Haemolysis and hence the concentra- ticularly critical. For heparin-dependent tions of free haemoglobin and potassi- methods (thrombin time, APTT), it is um, as well as the activity of lactate de- recommended that an amount of blood hydrogenase in plasma obtained from equivalent to twice the volume of the conserved blood, increase with the age catheter be discarded; the blood first of the transfused conserved material. taken after this should be used for non- haemostaseological investigations and Contamination of laboratory samples the subsequently obtained citrated blood by infusion solutions is the commonest only used for determining heparin- and often the most relevant form of insensitive analytes: Prothrombin time, preanalytical interference in the reptilase time, fibrinogen according to hospital (228, 242) (Tab. 6-1 ). Clauss, AT III, fibrin monomers. It is important that before transferring blood Blood should never be collected proxi- to the sampling vessel containing sodium mal to the infusion site. citrate solution there is no lengthy pause during which the blood in the catheter Specimens should be collected from the is allowed to “stand“. opposite arm. A certain period of time should be allowed to elapse following Mental stress infusion therapy (Tab. 6-2 ) The importance of mental stress on laboratory results is frequently under- Tab. 6-2 Recommendations for scheduling infusions and blood sampling estimated (anxiety prior to blood sampling, preoperative stress, etc.). Incre- Infusion Earliest time of blood ased secretion of hormones (aldoste- sampling in hours after rone, angiotensin, catecholamines, cor- cessation of infusion tisol, prolactin, renin, somatotropin, TSH, Fat emulsion 8 vasopressin) and increased concentra- Carbohydrate-rich solutions 1 tions of albumin, fibrinogen, glucose, Amino acids and protein hydrolysates 1 insulin, lactate and cholesterol have Electrolytes 1 been observed.

It is recommended that the laboratory Fig. 6-1 be informed of when and what type of infusions were carried out and when blood samples were taken.

Sampling from catheters If samples are to be taken from intravenous and intraarterial infusion catheters, the cannula should be rinsed with isotonic saline commensurate with the volume of the catheter. The first 5 mL of blood should be discarded before a blood sample is taken.

17 Sampling in the supine or upright position?

Posture lecular weight compounds show no change in their apparent concentrations It is a well-known fact that body posture when changing from the upright to the influences blood constituent concentra- supine position. As osmolality is mainly tions. This is caused by different me- mediated by such compounds, the first chanisms. First, the effective filtration is only modestly affected by changes in pressure (e.g. the difference between plasma volume (1–2%). Because of capillary pressure and colloidal osmotic partial protein binding, the concentra- pressure in plasma) increases in the tions of free and bound calcium are lower extremities when changing from affected in a different manner. Whilst the supine to the upright position. As a the concentration of free calcium is consequence, water is moved from the independent of posture, total calcium intravasal compartment to the interstitium; increases by 5–10 percent when this reduces the plasma volume by changing from the supine to the upright about 12 percent in normal individuals. position (172). Other changes are due Blood particles with a diameter of more to altered blood pressure which in turn than 4 nm are restrained by membranes causes secretion of vasoactive com- and cannot follow this volume shift. A pounds. In addition, the metabolic con- change from the upright to the supine sequences of regulatory changes due to position leads to a decrease in the effec- postural changes may alter body fluid Fig. 7-1 tive filtration pressure and hence to a vol- composition. Increase (%) of plasma ume shift in the reverse direction (176). The effects of posture on analytes in concentration of A change in plasma volume leads to venous anticubital blood are shown in various analytes when an apparent concentration change in Fig. 7-1. As expected from the described changing from supine to an upright position cells, macromolecules and protein- mechanism, most cellular and macro- (55, 131, 232, 239) bound small molecules. Most low mo- molecular analytes decrease between 5 and 15% compared to the supine po- 10 20 % increase sition. These effects can be more pronounced in patients with a tendency to haemoglobin leucocytes edema (cardiovascular insufficiency, liver haematocrit cirrhosis). Reduction in plasma volume erythrocytes induces a decrease in blood pressure total calcium which in turn leads to increased aspartate aminotransferase secretion of renin, aldosterone, norepin- alkaline phosphatase immunoglobulin M ephrine and epinephrine. A fall in blood thyroxine pressure causes decreased secretion of immunoglobulin G atrial natriuretic peptide which leads to immunoglobulin A albumin decreased plasma concentrations (211). total protein An example of the metabolic changes apoprotein B brought about due to posture is the cholesterol LDL-cholesterol urinary excretion of calcium which in- triglycerides creases during long-term bed rest (see HDL-cholesterol apoprotein AI Fig. 12-2, p. 28). 50 % increase

aldosterone Tourniquet epinephrine renin What happens when a tourniquet is norephinephrine kept on during sampling? A tourniquet

18 Effects of posture and tourniquet is usually applied to facilitate finding alanine aminotransferase the appropriate vein for venipuncture creatine kinase bilirubin (see p. 20). Using a pressure below the lactate dehydrogenase systolic pressure maintains the effective albumin alkaline phosphatase filtration pressure inside the capillaries. total protein cholesterol As a consequence, fluid and low mo- triglycerides lecular compounds are moved from the aspartate aminotransferase calcium intravasal space to the interstitium. erythrocytes haemoglobin Macromolecules, compounds bound to haematocrit protein and blood cells, do not pene- uric acid sodium trate the capillary wall so that their con- potassium centration apparently increases while chloride carbon dioxide the concentration of low molecular sub- creatinine urea stances is unchanged. leukocytes Fig. 7-2 shows the changes of different inorganic phosphate glucose analyte concentrations (93). The altera- –4–2 0 2 4 6 8 10 12 % tions of low molecular weight analytes observed following 6 min. of constric- portant prerequisite. The following con- Fig. 7-2 tion are in the range of ±3 percent and ditions should, whenever it is possible, Change (%) in serum therefore within the range of analytical be fulfilled: a preceding phase of rest concentration of imprecision. However, it has been shown and fasting, the same body position, various analytes after that constriction of the forearm muscles daytime and tourniquet application a tourniquet applica- causes an increase in serum potassium time, avoidance of repeatedly clench- tion time of 6 min (93) concentration. Therefore, during veni- ing and unclenching a fist. The tourni- puncture for potassium determination, quet application time should not be repeatedly clenching and unclenching longer than one minute. During a run- a fist should be avoided and a superfi- ning infusion the phlebotomy should be In comparing laboratory cial vein selected (45, 197). A venosta- performed on the opposite arm, in any results, reference inter- sis of two minutes did not change the case not proximal from the running in- vals should be obtained blood lactate concentration (mean fusion. If it is necessary to repeat sam- under identical condi- +2.2 %), but decreased the pyruvate pling for any other reason, the phle- tions with regard to blood concentration significantly (mean botomy should also be performed on body posture (176). –18 %) (108). The extent of changes in the other arm (176, 177). high molecular weight analytes depends on the duration of constriction. Fig. 7-3, for example, demonstrates total protein lactate dehydrogenase Fig. 7-3 changes of lactate dehydrogenase ac- (g/L) (U/L) Change in total protein tivity and total protein concentration. concentration ( ) and 90 The greatest effects are observed within lactate dehydrogenase activity ( ) in serum 5 minutes, little change taking place 85 120 thereafter (58). Constriction times of one during a 15 min minute with subsequent release of the 80 tourniquet application time (58) tourniquet have no consequences on 110 plasma serum analyte concentrations 75 and coagulation factors. To reduce the intra- and interindividual 70 100 variance of laboratory results a stan- dardised sampling procedure is an im- 0 2 5 10 15 min

19 What site for sampling blood?

The site of collection such as vein, ● Disinfection: Clean the venipuncture site. capillary or artery has a bearing on ● Vein Exposure: Apply a tourniquet. the procedural aspects of specimen col- ● Puncture Collection: See Fig. 8-1. lection (205). ● Mixing: Mix the blood in tubes Specific NCCLS documents address pro- containing additives or clot activators. cedures for the collection of blood spe- ● Prevention of Bleeding: Apply gauze to cimens by venipuncture (156), skin punc- the venipuncture site while removing ture (157) and arterial puncture (158). the needle. Apply a bandage to the patient’s venipuncture site. Phlebotomy ● Disposal: Dispose of the needle in a safety disposal unit. Critical steps in phlebotomy involve not The order of collection of tubes is im- only preparation of the patient for portant when multiple tubes of blood blood collection and proper collection are collected (Tab. 8-1 ). of the specimen, but also other steps amongst which are proper identification Quality of sample: of the patient. This avoids patient mix-up Examine to see whether the tubes are and ensures patient safety. Fig. 8-1 overfilled or underfilled. visualizes the steps to be taken in blood collection using evacuated tubes. Overfilling tubes intended for haematol- ● Identification: Match the patient's ogy determinations can cause spurious test order form with the patient number, results due to the absence of a bubble bar code or wrist band number. at the very top which will prevent prop- ● Position: Position of the patient er mixing of the specimen on rocker (sitting or recumbent) for venipuncture. type mixing devices (166). ● Materials: Ensure blood collection For certain procedures, the anticoagu- equipment (needle, collection holder, lant to blood ratio is critical (see p. 52). tubes) is at hand. ● Fig. 8-1 Inspection: Inspect patient’s arm, If multiple blood collection tubes are to Steps in blood select a vein while the patient’s fist is be collected (156), the following order collection clenched. of collection is recommended:

Penetrate the skin, hold- The blood is drawn up abReverse the positions of c ing the complete unit the hands as soon as the by the vacuum and flows between the thumb and needle is in place. Press into the tube (*) on its index finger of the right the tube home with the own. Release the tourni- hand. thumb of the right hand, quet immediately with the index and center your left hand still hold- fingers being supported ing the holder.

o on the flange of the 15 holder. Withdraw the tube with Agitate the tube very If multiple blood samples d the right hand, support- efgently, turning it over are to be taken, insert a ing the thumb on one of several times to ensure second tube and repeat the flanges of the holder. proper mixing with the the operations starting anticoagulant. from b.

* If the blood fails to flow into the tube, the needle should be revolved to exclude that it is occluded against the wall of the vein. If this does not result in blood flow, it has not found a vein. Before withdrawing the needle, disengage the tube so as to preserve the vacuum for further use. Recommence operations from a. Note: The phlebotomist in above steps (b to f) is wearing gloves

20 Phlebotomy, arterial puncture and sampling from catheters

Tab. 8-1 Recommended sequence of collecting dead space of the secondary tube] + various blood specimens dead space of primary tube. This formula assumes that 50 % of blood vol- 1. Blood culture Blood ume can be used as analytical volume 2. Non-additive tube Serum (71, Annex). 3. Citrate Plasma 4. Heparin Blood Sampling from artery 5. EDTA Blood/plasma Particular care should be taken when 6. Glycolytic inhibitor Glucose/lactate blood is collected from an artery (158). The common sites of arterial puncture If a blue stoppered citrate tube intended are the femoral artery, brachial artery for coagulation testing is to be drawn and radial artery. Other sites include scalp first, a 5 mL discard tube should be filled arteries in infants and the umbilical arter- to eliminate possible thromboplastin con- ies during the first 24 to 48 hours of life. tamination from the venipuncture site. Arterial puncture is necessary when venous blood does not permit the mea- How much blood is needed? surement of the relevant concentration The volume of blood collected from a of the desired analyte (e.g. blood gases, patient should be kept to a minimum to pH). Arterial puncture can be performed avoid blood loss that could render the either singly by inserting a short- patient anemic. The term investigational beveled, sharp needle into an artery. A anemia has been coined to refer to syringe can be attached to the needle blood loss due to repeated venipunctures. either directly or by way of tubing with Often, too much blood is drawn as is an adapter such as in a winged infusion apparent from a Mayo Clinic study set. Arterial blood can be collected where it was reported that for routine without suction when using 23-gauge collections, an average of 45 times the or larger needles, allowing the pressure required volume of specimen (range 2 in the artery to force the blood into the to 102 times!) was withdrawn, while for syringe. microcollection an average of 7 times the required volume of specimen (range Sampling with catheter 1 to 20 times) was withdrawn (40). In Continuous or repeated sampling of an earlier study it was reported that arterial blood can be performed by 47% of patients who required blood leaving a permanent needle, cannula transfusions had a phlebotomy-related or catheter in the artery or central vein. red blood cell loss of more than 180 mL, Care should be taken to ensure that no an amount equivalent to 1 unit of clot is formed at the tip or in the lumen packed red blood cells (198). of the catheter. Between samples, an It was recommended (71, Annex) to anticoagulant (preferably heparin) collect twice the amount of blood need- should be used to flush the needle. ed for analysis. The amount of blood When sampling with a venous catheter, needed may be calculated from the an- the coagulation tube containing sodium alytical portion needed, the dead space citrate should be filled before a he- of the analytical system and the sample parin-containing tube is collected. The tube using the following formula: first few mL of blood, representing 1 to Blood volume needed = 2 x [number of 2 volumes of the catheter, should be repetitive tests x (analytical volume + discarded to avoid contamination with dead space of the analytical system) + anticoagulant.

21 Blood from the skin

Fig. 9-2 Skin puncture is the procedure of choice Genie™ lancet if a small amount of blood is to be taken 13convenient, push-button ▼ from a pediatric subject. In adults, automatic operation blade capillary blood is used for blood gases, retraction glucose and lactate. There are differences between capillary and venous blood, 2 especially in oral glucose tolerance single use testing. 4 safe, uniform Blood obtained by skin puncture is penetration composed of a mixture of blood from depth the arterioles, venules and capillaries; it may also be diluted with interstitial and intracellular fluid. The depth of incision made to an infant’s heel is critical since puncturing deeper The relative composition of skin puncture than 2.4 mm on the plantar surface of blood will depend on variables such as the heel of small infants especially can blood flow to the skin during collection. damage the calcaneus or heel bone. Warming the puncture site prior to This can be avoided by using semi- blood collection in effect arterializes automatic disposable lancets (Fig. 9-2). the blood in the skin (157). After selection of the skin-puncture site, the site should be cleaned with a 70% Sites for blood collection are illustrated aqueous solution of isopropanol prior in Fig.9-3. They include the palmar sur- to puncture (125). After drying the site face of the distal phalanx of the finger with a sterile gauze to ensure that and the lateral or medial plantar surface residual alcohol has been removed of the heel (157). (since it would otherwise cause haemolysis), skin puncture should be per- Finger puncture should not be performed formed with a disposable lancet. The on infants since there is a likelihood of use of other disinfectants to clean the injuring the bone. There is a linear rela- puncture site should be avoided as this tionship between the volume of blood would spuriously elevate the results for collected and puncture site penetration uric acid, phosphorus or potassium (225). depth (7). Therefore, the lancet should be selected according to the site punc- The first drop of blood that flows after tured and the amount of blood needed skin puncture should be discarded by (Fig. 9-1). wiping with gauze, since this drop is

Fig. 9-1 Penetration depth of Genie™ lancet

1.25 mm 2.25 mm 2.0 mm1.0 mm 1.5 mm

22 Capillary sampling

Fig. 9-3 YES Capillary blood collec- YES tion with a typical micro collection device NO YES a) Assemble material and prepare patient. b) Select site, warm, disinfect and air dry (see examples of sites above).

c) Perform the puncture: Holding with firm grip, apply the Genie™ Lancet. d) Wipe away the first drop of blood. Position tube at puncture site and chan- Depress the plunger and release. Remove lancet and dispose into a con- nel blood flow down the center of the collector into the tube. Do not tainer for sharp objects. squeeze the area.

250 500 MAXI 500 500 250 MINI 500 250 250 EDTA e) Fill EDTA tube between 250 µl and 500 µl marks. f) Push down closure until a “click” is heard. g) Immediately mix sample by inverting tube 10 times. Do not shake. likely to be contaminated with tissue flu- for blood gas analysis should be made ids. By application of gentle pressure after warming the puncture site with a but without milking or massaging the towel soaked in running water at a area around the puncture site, free temperature not greater than 42 °C to flowing drops of blood should be col- bring about arterialization of the punc- lected by touching the drops with the ture site. The capillary tubes should be tip of a collector top and letting them free of air bubbles after collection. flow by capillary action into a properly On conclusion of specimen collection, labeled microcollection device. the puncture site should be pressed with a sterile gauze or swab and held Fig. 9-3 illustrates a typical microcollec- in place until bleeding stops. As a tion technique. In the event that drops safety measure, it is advisable not to do not flow freely from the collector top apply adhesive bandages over the into the microcollection tube, the tube puncture site of infants and children, may be gently tapped to facilitate the not only out of concern that the adhe- flow of drops of blood into the tube. sive may cause irritation but also due Upon completion of blood collection, the to the fact that the bandage could be- tube should be firmly secured. Additive- come loose and be swallowed by the containing tubes should be well mixed child. after specimen collection by gently inverting the tube approximately 10 times. The safety lancets used for skin puncture should be deposited in an appro- Collection of blood specimens into priate puncture-resistant safety con- heparinized capillary tubes intended tainer intended for such a purpose.

23 Did the lab mix up my sample?

Everyone concerned with diagnostic this number is printed together with the procedures is aware of the problem of name on any request, sample and sample identification. The mixing up of report. Ward, room and bed numbers Fig. 10-1 patients’ names, requests, samples or test may be of additional help. This is espe- Information flow results can have serious effects on patient cially useful if sampling is not done by during a laboratory treatment and should therefore be con- the same person as the one ordering test in a hospital. sidered as maltreatment. This chapter laboratory tests. HIS = hospital information system briefly summarizes the present state of the LIS = laboratory art of patient and sample identification Since such a large amount of information information system during the preanalytical phase (18). cannot be entered on small labels, a separate request form usually contains

doctor orders lab tests all the basic information together with the test orders linked to the sample by phlebotomy at ward a number or code only. pre-identified adhesive-backed patient sample labels Fig. on the back of the above text illustrates an example of a request form con- identified sample Ward HIS taining a sufficient number of labels for Lab printing accessiom number on all possible samples, used on sample adhesive-backed labels tubes (Fig. 10-2). Alternatively, a package results LIS patient sample identified and provided with of preprinted, self-adhesive labels with accession number work list the patient identification may be provid- edition ed on admission to hospital (Fig.10-3).

results entered technique with manual by a VDT reading of identification Techniques of identification Upon arrival in the laboratory, the patient As a minimal require- Name or number? has to be identified from the information ment the sample provided. This can be done visually by entering the lab should When admitted to a hospital, a patient reading the name and ward from the contain the following information: has to be identified by many people label and request form, transferring this including nurses, doctors, technicians information to the laboratory chart and • Name, prename and other persons. The same is true for all subsamples. The latter requires the • Date of birth any sample taken from the patient and generation of a sub-numbering system • Identity number of transferred to an in-house or external which is linked to the individual and sample, or patient or laboratory. As can be seen in Fig.10-1, the day of analysis (usually no more request communication between all parts of the than three digits long). • Ward number (sender, diagnostic process requires transfer of name of ordering this patient identification. A name used Many institutions, however, use electron- physician) to identify an individual has proved to ic means to transfer identification data: • Time of sampling (day, be insufficient in transferring the identity. this can be done on-line using a hospital hour, min) Name, prename and date of birth is information system which transfers the the most frequently used combination basic data of the patient together with of information for identification. In order the request directly to the laboratory to reduce the amount of information computer. In this case, the samples ar- involved, a patient number is allocated riving in the laboratory have to be in many hospitals. This number cannot be linked to a given request by bar-code used for any other individual. Ideally, or alternative code system fixed to the

24 Techniques of sample identification

Fig. 10-2 hospital admission ward Request number on adhesive labels for surgery 06 85 sample tubes John Smith 0001 ▼ ▼ Gluc UR CH

Fig. 10-3 Patient’s label provi- laboratory workstation laboratory reception sion at hospital admis- accession sion and their use in number the laboratory ▼

samples. In the example given in Fig. each analyzer used. This kind of identi- 10-2, the number on the sample is iden- fication is called indirect identification tical to the number of the request-form (by location) (128). Whereas direct iden- which is then electronically linked to tification mechanisms allow the sample the patient by the laboratory computer to be identified at any position and any (Fig. 10-3). time, indirect identification by position can be done only with the aid of a po- Recently, more sophisticated systems sition list or a computer system. In addi- have been suggested and will be widely tion, any sub-sampling has the inherent used in future: a two-dimensional bar danger of additional sample mix-up. code (Fig. 10-4), or transponder-chips fixed to the sample can contain all the ● Any sample should be identified un- necessary information including re- equivocally before being analyzed. quests and patient information. ● Direct identification procedures from primary samples should be used in preference to indirect identification Direct versus indirect sample and subsampling, to reduce misiden- identification in the laboratory tifications. ● Any sample whose identity and ori- The identity of the patient is unequivo- gin is not sufficiently documented cally linked to the sample container at should be processed to a stable form the collection point of the sample, pro- (serum, plasma, closed in refrigera- viding the primary sample is maintained tor) and missing information added throughout the analytical process. This before analysis is performed. needs serum/plasma separators to keep the analytical sample (plasma/serum) in the primary sample (blood) tube so that it can be identified directly by the reader of the analytical instrument.

If such a reader is not available or subsamples have to be distributed, a sampling device may be used to automatically Fig. 10-4 Two dimensional bar-code in compari- define the positions of each sample in son to unidimensional bar-code.

25 A precious sample

How to collect CSF? (50 mL/day in adults). Particularly when searching for tumor cells, it is important Puncture sites to obtain as much CSF as possible (up CSF and serum form an inseparable unit to 30 mL being optimum in adults) (Tab. 1 Fig. 11-1 in modern diagnostics; for this reason 11- ). The sampling time should be as Sampling of CSF and serum should be collected as long as possible, using a needle as fine cerebrospinal closely together as possible (103). The as possible, to avoid headache follow- fluid puncture site is generally lumbar, but also ing puncture.

Which specific aspects of CSF sampling are important?

The fasting patient should be seated or asked to lie on his/her side on a flat support. The patient‘s back should be bent forwards and the position secured by an assistant. The musculature should be as relaxed as possible (Fig. 11-1). The time at which the sample is taken should be noted, together with information on any initial treatment (e.g. in bacterial meningitis).

It is recommended that an “atraumatic“ pencil-shaped needle (0.7 mm outer di- ventricular, suboccipital or via a shunt ameter), as designed by Sprotte and and should be noted. Whitacre (Fig.11-2), be used instead of a 22G needle with a Quincke cut so as The puncture site should be marked, to avoid post-puncture syndrome and the area disinfected. Treatment of (headache) (24). weals with a local anesthetic is desir- able for the patient. Puncture should be In order to avoid contamination CSF saggital and sloping upwards (20 °) should be obtained and transported in (Fig. 11-1). closed tubes.

The CSF should be placed, under aseptic Amount required conditions, in separate colorless poly- The quantity taken depends on the styrene tubes with stoppers (sterile for clinical situation and is not so critical in microbiology and dust-free for cytology adults; CSF is very rapidly replenished or clinical chemistry). For cytology, the use of additives such as EDTA and fluo- Fig. 11-2 ride should be avoided. After a sample “Atraumatic” pencil – has been obtained, the needle should shaped needle be removed and the wound covered with a plaster. Patients should be kept lying on their stomachs for at least 30 min to avoid subsequent leakage.

26 Cerebrospinal fluid (CSF)

Tab. 11-1 Sequence and quantity of CSF are stable for 4 to 6 days at room tem- recommended perature or – when fixed in acetone – for 3 to 12 months at –70 °C (immuno- Fraction Adults Infants cytology depending on the antigen) Discard the first 0.5 mL and all (237). artificially sanguineous CSF Microbiology* ~ 2 mL ~ 1 mL Cytology > 10 mL > 1 mL Special hints for CSF sampling Supernatant used for (tumor cells!) (tumor cells!) ● Sampling in several portions means clinical chemistry that the concentration gradients that Total 12 mL 2 mL always exist (e.g. for albumin and IgG) *Before commencing chemotherapy, or 2–3 days after its withdrawal. are not taken into account. This can be avoided if the entire volume is first collected in one tube, well mixed and then What about storage and transport? divided into aliquots. Most important of all: As soon as possible after the sample ● Gloves dusted with talcum powder has been taken, it should be sent to the should not be used when withdrawing laboratory by courier. CSF, as CSF cytology is thereby invali- dated. CSF is not particularly “cell friendly“. Transport in an icebath over 200 km by ● The presence of up to 6000 leuko- car (approx. 3 hours) is possible. Sta- cytes/µL should not alter the lactate bility data of individual components concentration within 3 hours at room are given in the Annex. Further recom- temperature (120). mendations are given in Tab. 11-2 . ● Deep-freezing of CSF is better carried out at –70 °C than at –20 °C since, for Tab. 11- 2 Recommendations for transport and example, oligoclonal protein bands storage of CSF slowly disappear after storage over Up to 1 hour Do not cool 6 months at –20 °C. Up to 3 hours Transport on ice Never deep freeze No additives No partial fixation Long-term storage After centrifuging off cells, immediately cool to –70 °C in glass or polypropylene vessels that can be tightly closed.

For cytological investigations, instead of original CSF, send cytocentrifugation preparations to the laboratory. This should be prepared close to the patient using a special centrifuge (centrifugation for 20 min at 180 g). These samples

27 A sample that is nearly always available

Urine should be avoided. The different kinds A review of the ten most popular errors of sampling are given in Tab. 12-1 . in the analysis of urine revealed that For pediatric and newborn patients, some of the problems were of a prean- urine specimen collection bags with alytical nature: samples were too old, hypoallergenic skin adhesive should be sampling vials had been contaminated, used. First, the pubic and perineal ar- the samples were not homogeneous eas should be cleaned with soap and and preanalytical factors had not been water. Then, the adhesive should be adequately taken into account when in- pressed all around the vagina or the terpreting test results (59, 109). The qual- bag fixed over the penis and the flaps ity of the collection vial is a critical fac- pressed to perineum. The container tor in this context. The ideal container should be checked every 10 –15 min for any urine specimen is a wide-mou- (153). Urine samples obtained in the thed bottle of appropriate size (Fig.12- morning offer several advantages. A 1). Urine containers used should either high degree of osmolality indicates an be disposable or, if not, detergents intact concentrating ability on the part have to be reliably removed prior to of the kidney. Urine samples obtained

Fig. 12-1 use. If the urine is to be analyzed bacte- in the morning are particularly useful in Containers for urine riologically, containers have to be ster- identifying mycobacteria. Deviations specimens for qualita- ile. In protein and hormone analysis, due to diet, physical activity and pos- tive and quantitative analyte absorption to the vessel wall ture are diminished. Fig.12-2 shows the testing

% 240

200

160

percent of excretion 120

100

immobilization

Tab. 12-1 : Different types of urine specimens and their use in the laboratory

1. Random or spot urine Qualitative chemical determinations -4-20246810 2. First morning urine Cellular constituents and casts weeks ▲ 3. Second morning urine Quantitative determinations related Fig. 12-2 (7 – 10 a.m.) to creatinine Urinary excretion of calcium during a six-week 4. 24 h urine Quantitative determinations immobilization period

28 Urine and saliva as diagnostic probes

Fig. 12-3 ammonia Influence of 4-day urea starvation on urinary creatinine excretion of elec- 212

sulfate 144 % changes trolytes and inorg. phosphate substrates. Change (%) during ( ) and one chloride 100 day after the magnesium voluntary starvation calcium 50 ( ) related to the potassium starting value (106) sodium

-50

-100 increase of the urinary excretion of cal- mended for bacteriological investiga- cium to 240% during a six week immo- tions (59, 109). bilization period (240). In Fig.12-4, the collection of a mid- Fig.12-3 gives an example of the influ- stream urine (clean-catch urine) is ence of diet on the urinary excretion of shown schematically. Mid-stream urine electrolytes and some substrates. Under containing bacteria >105/mL is consid- these special conditions, five healthy ered to indicate significant bacteruria. volunteers received only distilled water Analysis of urine samples should ideally during a four-day voluntary period of proceed within one hour following starvation. Most analytes showed a re- sample generation (except in the case duced level of excretion. Only ammonia of 24-hour urine samples). A short pro- excretion is increased as a consequence cessing time is of particular importance of the metabolic acidosis induced by once a morphological evaluation of the starvation (106). labile urinary sediment constituents has to be performed. Thus, the stability of For quantitative analysis, one should erythrocytes and leukocytes in urine is Fig. 12-4 use timed urine, preferably in the form dependent on both pH and osmolality. Collection and of 24-hour urine collection (104). Mid- A pH above 7.5 and an osmolality sampling of mid- stream catheterized or suprapubic below 300 mosm/kg results in rapid stream urine (clean- puncture urine specimens are recom- degradation of these cells (165). Other catch urine)

29 A sample that is nearly always available

Fig. 12-5 albumin urea Influence of storage citrate uric acid time on the recovery creatinine calcium of various analytes in glucose magnesium urine samples without oxalate inorg. phosphate additives and storage t-protein potassium at room temperature sodium (% changes from the starting point) (186) % decrease

2 days

4 days

6 days 100 analytes tend to form crystals at physio- No single additive is available that stabi- logical pH (calcium, oxalate, uric acid) lizes all classes of compounds. or are decomposed if not adequately stabilized (glucose, urea, citrate). For quantitative determination, storage Fig.12-5 shows the changes (%) of with a stabiliser at room temperature is various analytes after a storage of two, recommended. If the urine samples are four and six days at room temperature stored at 4–6 °C for calcium, magne- without additives. Citrate, glucose, sium, oxalate and phosphorus determi- oxalate and magnesium are unstable. nation, acidification (pH 1.5–2.5) and, Addition of sodium azide in a final for uric acid, alkalization (pH > 8.0) is concentration of 10 mmol/L urine for necessary. For special analytes, differ- the same time and temperature stabi- ent types of preservatives are neces- lized all these constituents for 6 days at sary (see Tab. 12-2 ). room temperature (186). Tab. 12- 2 lists a number of additives which are used to stabilize compounds in urine. Saliva Saliva can be used for analyzing various compounds such as steroids Tab. 12-2 : Urine preservatives (59, 91) and drugs. Samples obtained can be Preservative Analytes stabilized derived from one gland only (gland- specific saliva) or be a mixture of Thymol, 5 mL of a 10% solution products from different glands (mixed in 2-propanol Most constituents saliva) (Fig. 12-6). The latter specimen Sodium azide, 10 mmol/L urine Glucose, urea, uric acid, citrate is used for routine procedures. Different potassium, calcium, oxalate techniques are employed to obtain saliva Hydrochloric acid, 25 mL 6 mol/L per Catecholamines and metabolites, from the oral cavity (77). Several collec- 24-hours urine volume 5-hydroxyindolacetic acid, tion devices have been developed to calcium, magnesium, phosphate standardize the collection of saliva for hormone and drug monitoring. Tab. Sodium carbonate, 2 g/L urine Porphyrins, urobilinogen 12-3 provides a selected listing of the Urine pH > 8.0 Uric acid presently available devices and adsorb-

30 Urine and saliva as diagnostic probes

Tab. 12-3 : Commercially available saliva collection devices using adsorbing materials Name Manufacturer Adsorbing material Sampling device Salivette Sarstedt Cotton roll with citric acid or polyester roll Centrifugal device Omni-Sal Saliva Diagnostic Systems Adsorbing pad with fluid volume indicator Separation by pressure through a filter into a buffer containing tube Orapette Trinity Biotech Rayon ball sampling Expulsion by screwing a piston Ora Sure = Epi Screen Epitope Pad on "lollipop” stick Centrifugal tube with antimicrobial buffer Accu Sorb™ Avitar Technologies Polymer pad fixed on the closure Squeezing manually (milking) into a plastic tube Oral Screen Avitar Technologies Polymer foam cube sampling Squeezing by pressure on the surface Abusa-stick Chem-Elec Saliva swab Alcoscan Lifescan Saliva swab Q.E.D. (used SCT Technologies Cotton swab on a stick Squeezing by pressure on the for saliva alcohol test) surface

ing materials. No one device can be considered ideal for all purposes. Devi- ces using materials adsorbing saliva are either contaminated with substances interfering with the measuring procedure or the drug itself is adsorbed by the materials to varying degrees depending on the device used. Recoveries of drugs have recently been reported to range from 59 to 107% (78).

Saliva has several advantages in drug monitoring compared to blood. Goro- discher has reported that 85% of pa- rents and 50% of children indicated a preference for saliva sampling over venous blood withdrawal (64). The ease Fig. 12-6 Collection of saliva from different locations of the oral cavity. In the of collection of saliva makes it ideal for upper part 4 cotton rolls connected by a thread were placed above the self-testing at home and in cases where tongue. At the bottom 3 shortened cotton rolls connected by a thread were blood sampling is difficult (e.g. in new- placed below the tongue. On the left and right side metalline strips are borns). Limitations in sampling posed shown which are wrappped in polyethylene foil with a rectangular opening by saliva are related to viscosity and in in the area of the orificium of the ductus parotidis. The 4 parts were placed into the oral cavity and left there for 9 minutes without moving the tongue. some cases to the difficulty in obtaining Saliva was collected from the strips and the cotton rolls by centrifugation in sufficient volume. The saliva/plasma ratio Salivettes® (78) of drugs of abuse has been reviewed (78).

31 Plasma or serum?

Serum is obtained from whole blood by pling. Anticoagulants inhibit clot forma- centrifugation after completion of the tion through various mechanisms (72, platelet and clotting factor coagulation Annex). The required concentration of Blood process. Serum must therefore be re- anticoagulants and their composition is

Anticoagu- No anticoag- garded as an artifact. By definition, described in an international norm lants ulants it is devoid of clotting factors but is (89). can be store for enriched with the cellular components centrifuged 30-–45 min of platelets and metabolic products. For some analytes, there are diagnosti- immediately undisturbed cally relevant differences between the and,if possible, in the dark; Plasma is the virtually cell-free super- results obtained from serum and those centrifuge natant following centrifugation of whole obtained from plasma (see Tab.13- 1 Plasma Serum blood, the coagulability of which is and Fig. 13-1). A linear correlation has inhibited by the addition of anticoagu- been shown between the difference in lants during or immediately after sam- serum-plasma potassium and phosphate and the number of platelets in Tab. 13-1 : Analytes with diagnostically relevant serum/heparinized blood (Fig.13-2). Some constituents plasma concentration differences and their main causes (226) found in high concentrations in platelets Analyte % change in comparison Main cause of the serum/ can therefore not be correctly deter- to the mean in plasma plasma difference mined in serum, e.g. acid phosphatase Potassium +6.2 Lysis of the cells, particularly activity, neuron specific enolase, dopamine and serotonin (69). the platelets* Inorganic phosphate +10.7 Release from cellular elements Differences between the values ob- Total protein –5.2 Effect of fibrinogen tained in serum and plasma are due to Ammonia +38 Thrombocytolysis, hydrolysis of the following physiological and technical glutamine reasons: Lactate +22 Release from cellular elements ● The analyte may be consumed during * Also causally responsible for high potassium values in serum are – in addition to thrombocytolysis – haemolysis clotting: fibrinogen, platelets, glucose. and extreme leukocytolysis (when leukocyte counts are > 50 G/L). Concerning the platelet influence in whole ● The analyte may be released from blood, the following applies: an increase in the platelet count by 100 G/L means an average increase of 0.11 mmol/L in the difference between the serum and plasma potassium values. cells during clotting: potassium, lactate dehydrogenase, phosphate, ammonia, Fig. 13-1 total protein lactate. Plasma serum differen- albumin ● The anticoagulant may interfere with ces obtained in 4 mL TSH the assay or contaminate some assays: separator tubes (226). transferrin Ratio of the median dif- ASAT creatine kinase ference between serum total bilirubin 1.8 and plasma and the sodium 1.6 coefficient of variation iron 1.4 of the analytical proce- cholineesterase 1.2 alkal. phosphatase dure used 1.0 ▲ direct bilirubin 0.8 triglycerides (mmol/L) higher in plasma higher in serum LDH diff 0.6 K Fig. 13-2 inorg. phosphate 0.4 Dependence of plasma- potassium 0.2 γ− serum difference in glutamyltransferase 0.0 triiodothyronine potassium -0.2 lactate on platelet count in -50 5 10 blood (121) serum – plasma 200 400 600 800 1000 1200 1400 1600 ratio of x 100 9 plasma x cv platelet count (x 10 /L)

32 Differences to be considered

γ-glutamyl transferase, lithium in flame region of the γ-globulins and can simu- When obtaining a photometry, when calibrated with lithium. late or mask an M-gradient. blood sample, it is im- 2. Method-dependent interference. Anti- perative that attention ● Methodology of the respective deter- coagulants can – as potential complex- be paid to ensuring mination, including for example the type ing agents and enzyme inhibitors – thorough mixing of the of measuring instrument used (mono- lead to method-dependent interference. blood with the anticoagulant; foaming should chromatic/bichromatic measurement) Every new procedure should therefore be avoided. (80) may cause interference. This in- be tested for anticoagulant interference. cludes interference of fibrinogen with 3. Cation interference. When heparina- Not more than 2 min- some heterogeneous immunoassays. tes are used, lithium or ammonium may utes should elapse be- interfere with the methods for deter- tween the beginning of mining them. stasis and the mixing of What advantages does plasma the blood with anti-co- have over serum? The annex summarises the present state agulant. The recommendations published by the of knowledge on the use of anticoagu- Working Group on Preanalytical Quali- lants in the individual analytes. ty (72, Annex) has described the advantages and disadvantages of using If serum or plasma separator tubes are plasma versus serum: used, renewed centrifugation following storage of the sample in the refrigerator 1. Time saving. Waiting for blood to should be avoided as this would lead clot is eliminated. The centrifugation to noticeable increases in the concen- period can be reduced considerably by tration of cellular constituents like potas- increasing the rotation speed. sium, inorganic phosphate and lactate 2. Higher yield. Approx. 15–20% more dehydrogenase. plasma than serum can be obtained from whole blood. 3. Virtually no interference due to sub- Types of plasma sequent coagulation. Post-centrifugal Presently various different types of plas- coagulation can occur in serum. This ma are recommended for individual effect does not occur in plasma. analytes and test procedures. Thus buff- 4. Results from plasma are more repre- ered sodium citrate is recommended for sentative of the in vivo state compared coagulation testing. Various types of plas- to serum (Fig. 13-1). ma obtained from citrated blood are 5. Lower risk of haemolysis and throm- used: bocytolysis. In healthy persons, free haemoglobin is about 10 times less Tab. 13-2 Different types of plasma concentrated in plasma than in serum. Plasma Relative centrifugal Centrifugation In plasma, the platelets remain intact in force (g) time (min) vitro; there is hence no pseudohyper- Platelet-rich 150–200 5 kalemia here, as is found in serum Platelet-poor 1000–2000 10 (121). Platelet-free 2000–3000 15–30

K -EDTA is recommended for haemato- What are the disadvantages of 2 logical cell analysis and analytes sensi- plasma relative to serum? tive to metalloproteinase degradation. 1. Protein electrophoresis is altered. Heparin plasma is recommended for Fibrinogen appears as a peak in the nearly all extracellular constituents.

33 Take a lavender tube!

Additives Heparin In addition to anticoagulants such as Heparin, convenient for use in blood EDTA, heparin, citrate and oxalate, other collection tubes, functions by accelerat- additives have been used for blood col- ing the inhibition of factor Xa by an- lection. tithrombin III. In contrast to low molecular weight heparin preparations, the To insure that there is no confusion on conventional high molecular weight the part of the phlebotomist in identify- heparin used in blood collection tubes ing the proper tube, the stoppers and also possesses antithrombin activity. closures of anticoagulant and additive- containing tubes are colour-coded. While the lithium salt of heparin is widely Thus, for anticoagulant-containing tubes, used to obtain plasma for clinical chemi- lavender is the stopper colour code for cal analysis, blood collection tubes con- EDTA, green is for heparin and blue is taining salts of sodium or ammonium he- for citrate. Glycolytic inhibitors such as parin are also commercially available. fluoride or iodoacetate either alone or Ammonium heparin may limit the blood in combination with an anticoagulant urea nitrogen assay when ammonium such as heparin or EDTA have been ions are being measured (144). colour-coded gray. Additional letter codes have been included in the ISO Fig. 14-1 EDTA Colour codes for anti- norm (89). coagulants and This compound functions as an anti- additives described Fig.14-1 shows pictures of tubes used coagulant by binding calcium. EDTA is by ISO 6710 (89) in blood collection and identified by discussed in the haematology chapter their proper colour code. (p. 54).

Tubes containing acid-citrate-dextrose Citrate (ACD A or B formulation) are used for Sodium citrate also functions as an anti- the preservation of cells and are colour- coagulant by chelating calcium. Sodium coded yellow. citrate is discussed in the coagulation chapter (p. 52).

Glycolytic inhibitors Tab. 14-1 Additives of colour coded tubes Both sodium fluoride and lithium Tube Application Colour/letter code iodoacetate have been used in blood 1. Plain (no additive), Clinical chemistry and serology red/Z collection tubes to preserve glucose. yields serum Mannose and fluoride have also been 2. Heparin (12–30 U/mL) Plasma chemistry green/LH or NH suggested. When combined with an anticoagulant such as Na -EDTA (1 3. K2- or K3-EDTA Haematology and selected chemistry lavender/K2E or K3E 2 (1.2–2.0 mg/mL) determinations on plasma mg/mL of blood) or potassium oxalate 4. Sodium citrate Coagulation light blue/9NC (2 mg/mL of blood), the nominal con- (0.105–0.129 mol/L) centration of fluoride used to inhibit glycolysis is 2.5 mg/mL of blood (60 5. Sodium fluoride Glucose, lactate gray/FX mmol/L). (2–4 mg/mL)/potassium oxalate (1–3 mg/mL)

34 Additives and colour codes

Fig. 14-2 Fluoride inhibits the enzyme enolase in rapid inhibiting mixture +NaF Preservation of the glycolytic pathway and thereby pre- glucose with glycolytic 100 vents the degradation of glucose (34a). inhibitors (49) NaF In contrast to fluoride, iodoacetate acts 90 on glyceraldehyde-3-phosphate de- 80 no inhibitor hydrogenase. After an initial loss of glucose during the first 3 hours of 70 blood collection (an average loss of 9 mg/dL in healthy subjects), both 60 fluoride or iodoacetate are effective in preserving glucose for at least 3 days glucose concentration (% of initial) 50 024202224(h) (34a, 193). It should, however, be recognized that blood specimens with a high storage time at room temperature white blood cell, red blood cell or platelet count will have more rapid Preservation of cells glucose consumption, before inhibitors such as fluoride or iodoacetate become Anticoagulant – additive mixtures such effective (34a). In newborns, particular- as ACD (anticoagulant-citrate-dextrose ly because of the high haematocrit, as or acid-citrate-dextrose) have been much as 68% of glucose can be con- used in blood collection tubes to pre- sumed in blood specimens collected serve red blood cells. ACD is available without glycolytic inhibitors and stored in 2 formulations, A and B. The differ- at room temperature for 5 hours (126). ence between the 2 formulations is the To overcome this problem, inhibitors blood to additive mixture ratio. which inhibit the first enzyme hexoki- nase in the glycolytic pathway have Tab. 14- 2 been suggested. Such an inhibitor is The composition of ACD formulations mannose, which is used in combination ACD A ACD B with fluoride to better preserve glucose Sodium citrate 2.2 g/dL 1.32 g/dL (117). The inhibition by mannose, which is a competitive inhibitor, is short-lived, Anhyd. citric acid 0.73 g/dL 0.44 g/dL inhibiting glycolysis only up to 4 hours Dextrose (glucose) 2.45 g/dL 1.47 g/dL after blood collection. However, be- ACD/blood (v/v) 1/5.67 1/3 yond 3 hours of specimen collection, pH 5.05 5.10 fluoride would have become effective; thus, a mixture of mannose (2 mg/mL In the ACD A formulation, an additive to of blood) and sodium fluoride (2 blood ratio of 1:5.67 is used whereas mg/mL of blood) would have been ef- in the ACD B formulation the additive to fective in minimizing the loss of glucose blood ratio is 1:3. which would otherwise have occurred had blood been collected using either ACD preserves red blood cells for 21 sodium fluoride or lithium iodoacetate days when blood is stored between 1 alone. and 6°C (22).

Fig. 14-2 depicts the efficacy of glycolytic inhibitors in preserving glucose (49, 220).

35 Fax me a sample

Effects of time and haematology or to perform blood gas temperature during transport analysis (80). If for some technical reason a long distance transfer of the sample is The transfer of samples to the laborato- required (e.g. by mail or laboratory ry can be accomplished in different courier), then whole blood samples ways. Normally, transfer times are short should be avoided. Fig.15-1 demon- when the laboratory is located close to strates the influence of temperature and or in clinics and represent no problem. duration of storage of clotted blood The time from drawing the blood sample samples on some target analytes (84, to centrifugation, however, should not 163). exceed one hour. Some analytical procedures require special additives Release of potassium from erythrocytes such as sodium fluoride/ oxalate for is minimal at room temperature due to quantification of lactate (9) or sodiumb- the temperature-dependent activity of the orate/serine EDTA for quantification of Na+, K+-ATP-ase. This effect increases ammonia (56). Determination of free both at 4°C and above 30°C. Glucose haemoglobin in plasma requires gentle concentrations decrease with increasing handling of the EDTA blood sample. temperature, whereas the opposite phe- Fig. 15-1 Transfer to the laboratory may proceed nomenon is observed with inorganic Temperature and time effect of storage of either by courier or a pneumatic tube phosphate because the activities of clotted blood without delivery system. State-of-the-art systems phosphatases in serum and red blood anticoagulant on of the latter type ensure gentle transfer cells increase this compound. As various serum ana- thereby avoiding haemolysis. Such demonstrated in Fig.15-1, duration of lytes (163). ( ) 4 °C, samples can be used to determine storage at a given temperature is an ( ) 23 °C, ( ) 30 °C target analytes in clinical chemistry, influencing factor. If a whole blood sample is stored for two hours at 23°C, glucose concentrations decrease by glucose 1000 about 10 percent (Fig. 15-1). 900 100 800 Pathological samples may show devia- 700 78 tions from the usually observed effects 600 of time and temperature. Time-depen-

mg/L 500 46 dent decrease of glucose in whole

400 change (%) blood samples is enhanced in leukocy- 300 29 200 tosis. Similarly, the time-dependent increase in ammonia is enhanced in sam- 024682448h ples with elevated γ-glutamyltransferase potassium activities (56). Antibodies may alter

11 252 haematological cell counts depending 10 on the temperature dependence of the 9 198 antibodies (see p. 78-79). 8 7 mmol/L 6 Many clinical chemical analytes such change (%) 5 as electrolytes, substrates or enzymes 4 100 are not affected by a mail transport 3 time of up to four days. The haemoglo- 024 6 82448h bin concentration and the erythrocyte count are also stable. Major differences

36 Samples: From the Patient to the Laboratory. W. G. Guder, S. Narayanan, H. Wisser, and B. Zawata Copyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-30981-8 Effects of time and temperature during transport

Fig. 15-2 leucocytes Stability of various alanine aminotransferase mean cell volume analytes during mail alkaline phosphatase haematocrit transport (13, 122) creatinine erthrocytes bilirubin haemoglobin lactate albumin dehydrogenase calcium γ-glutamyl- 6 transferase potassium 4 aspartate sodium aminotransferase 2

-2 changes (%) -4 -6 -8 -10

are observed for the haematocrit, the prothrombin time, APTT, thrombin time, mean cell volume and bilirubin (Fig. 15- protein C and factor V are stable for 2). A reliable examination of a differen- eight hours at room temperature, pro- tial leukocyte count requires prepara- vided that the plasma is not separated tion of the blood smear within three from the erythrocytes. This is not valid hours of sampling. The included Annex for factor VIII and protein S. The sample ”The Quality of Diagnostic Samples“ stability of patients with heparin thera- Fig. 15-3 Thrombin time (TT) de- may be used to find additional details py, stored at room temperature or in termination in plasma on analyte stability in full blood and the refrigerator is below eight hours for samples of patients serum/plasma. the global tests and the determination with and without he- of single coagulation factors. There- parin therapy, stored at Numerous studies related to the stabi- fore, all patient samples should be room temperature (RT) lity of clinical chemical analytes exist analysed within four hours at room tem- and 6° C (83) compared to the few investigations on perature after sampling. If this is not ( ) without heparin at RT the stability of haemostasiological para- possible, platelet poor plasma should ( ) with heparin at RT meters (83, 216). As shown by Heil et be stored at –20° C. ( ) without heparin at al. the stability of coagulation factors in 6° C patient samples is dependent on ( ) with heparin at 6° C whether they are on heparin therapy or 120 not. For the thrombin time this is clearly demonstrated in figure 15-3. The caus- 110 es for the changes of stability are 100 manyfold, i.e. loss of the haemoglobin buffering system after plasma separa- 90 tion from the red blood cells, an in vitro 80 fibrinolytic effect, or drug influences on 70 platelets. The results of the study (83) 60 can be summarised as follows: citrated blood samples of patients without he- Abweichung vom Ausgangswert (%) 50 parin therapy for the determination of 08 24 48 72 96 168 h

37 Samples in transit

Fig. 16-2 When blood or other body fluids from Infectious substance human subjects are mailed to a distant label for package laboratory, several safety regulations containing etiologic have to be complied with. In addition, agents

▲ the integrity of the sample has to be preserved to insure accurate analysis by the investigator. Specimens that are INFECTIOUS SUBSTANCE mailed must “withstand leakage of con- IN CASE OF DAMAGE OR LEAKAGE tents, shocks and pressure changes, IMMEDIATELY NOTIFY PUBLIC HEALTH AUTHORITY IN U.S.A. and other conditions incident to ordinary NOTIFY DIRECTION – ODC ATLANTA GA handling in transportation” (50, 159, 404/633-5313 196). 6

The persons dispatching diagnostic samples have to assure, that the contents are packaged in a way insuring required. The responsibility for the post- arrival in an undisturbed state. No risk ing of infectious materials is with the should occur for humans, animals and dispatching physician’s site. the environment during transport. In Europe the standard package EN A biohazard label as Regulations concerning transport by 829 (Fig. 16-1) is accredited (50). No shown in Figure 16-1 post are reported in national standards glass is allowed as sample material to should be affixed to the (43). Here samples with infectious sub- reduce the risk of breakage and possi- package. stances have to be treated differently ble harm to people involved in trans- compared to materials with a low risk port. of infection (like most samples of blood, serum, urine, stool, swabs, slides and The package for biological materials filter papers). For posting of diagnostic consists of the following parts: samples even if non volatile the package can be sent by letter post. Pack- ● The inner package for the sample ages with infectious materials have to material, be labelled with the remark: DIAGNOS- ● absorbing material, TIC SAMPLE/INFECTIOUS HAZARD.For ● the outer package ensuring speci- international traffic and posting men records and laboratory forms, Fig. 16-1 the description in French language: ● the box or mailing bag. Biohazard Label for ”Matières Biologiques Périsablés” is air-shipped specimens Instead of the outer package several specimen containers up to 500 mL may ETIOLOGIC AGENTS be packaged in one box consisting of card board, wood, suitable plastic or metal according to the regulations for BIOHAZARD biohazard transport.

Packaged in Complance with 42 CFR Part 72 Remarks: In any case where infectious IN CASE OF DAMAGE substances are contained in the pack- OR LEAKAGE, NOTIFY age, additional secondary container is CENTERS FOR DISEASE CONTROL needed to prevent any leak of material (404) 633-5313 by any mechanical challenge.

38 Legal standardization for mailing samples

Fig. 16-3 Always remove injection needles when Packaging of speci- mailing blood sampling systems. primary container mens for transport absorbent packing material according to NCCLS Package glass slides adequately to en- (159) sure they do not get damaged if cap knocked, dropped or if pressure is ap- secondary container plied. specimen record Ship stool specimens in leak-proof, cap (CDC 3.202) screw-capped containers.

EA label When mailing dried blood specimens shipping container on filter paper, place in a strong paper envelope and then seal in plastic-lined, padded post bag. This provides protec- address label tion against potentially infectious dried blood specimens and ensures the integrity of the specimens during transport.

For shipping frozen and refrigerated waterproof tape specimens, an insulating material such as a polystyrene container is adequate. culture Dry ice should be used for freezing. absorbent packing Caution should be taken to insure that material the container packed with dry ice is able to release carbon dioxide gas so cross-section of as to avoid a build up of pressure that proper packing could cause the package to explode.

NCCLS document H18-A2 describes information is found together with the procedures for the handling and trans- requirements and testing procedures report of diagnostic specimens and etio- garding transport packages, samples, logic agents (159). absorbing materials and protective vessels. The label to be used is shown in In Europe details are regulated by Eu- Fig. 16-4. ropean Standard prEN 829 for in vitro diagnostic systems (50). Here detailed

Fig. 16-4 ches Untersuch gisches Untersuchun Medizinis ungsgut Biolo gsgut Label to be used for transport of medical and biological specimen according to EN 829 (German version)

39 How to keep a sample ”fresh“

10 rules and some 6. Storage problems are reduced if recommendations disposable sampling systems are used.

1. The procedure is governed by the 7. Separating agents (e.g. gel sepa- stability of the constituents of the sample. rators) improve the serum/plasma The most important causes for altera- yields and enable serum to be left in tions to the quality of specimen are: the original tubes above the blood (111).

● Metabolism of the blood cells 8. Avoid shaking the sample vessels ● Evaporation/sublimation (pneumatic tube dispatch systems!): risk ● Chemical reactions of haemolysis. ● Microbiological decomposition ● Osmotic processes 9. Always store sample vessels con- ● Effect of light taining blood vertically; the clotting ● Gas diffusion procedure is accelerated.

2. Rapid transport and short storage 10. Label infectious material and handle times improve the reliability of labora- it with particular care. tory results.

3. Specimens and samples are preserved 8 special rules and some more longer the cooler they are stored (but useful recommendations note exceptions!). 1. Avoid storing of whole blood. Infor- 4. Specimens and samples should mation on sensitive analytes is given in always be stored in closed vessels the Annex (see enclosure). (evaporation!). Blood samples should reach the labora- 5. The danger of evaporation also ex- tory within 45 min of collection in order ists in refrigerators (condensation of to ensure that centrifugation and sepa- moisture on the cooling elements). ration of the sample is carried out within 1 hour (49, 112, 159).

2. Avoid glycolysis to keep glucose, Fig. 17-1 lactate and pH stable. Glycolysis can Formation of concen- 2.0 be avoided by the addition of an in- tration gradients of calcium in plasma hibitor in conjunction with an anticoag- samples after rethaw- 1.5 ulant (171, 220). ing without mixing 1.0 3. Avoid the effect of light otherwise

calcium mmol/L there will be a fall in the values of bilirubin, vitamin C, porphyrins, creatine 0.5 kinase (CK) and folic acid. upper

middle 4. Reduce contact with air as far as upper middle lower possible. If this is not done, evaporation/ lower sublimation will result in an apparent increase in the concentration/activity

40 Storage of samples in the laboratory of all non-volatile components. This is undissolved material and, if necessary, particularly the case when the volume bring into solution by careful warming. of the sample is relatively small and the surface area is relatively large. 8. Store samples after analysis in such a way as to permit the confirming of 5. Whole blood should not be stored in results, checking the identity of samples the refrigerator. When urine is cooled, or performing additional tests for salts may precipitate out of the solution medical or legal reasons: (calcium and magnesium phosphate, Tab. 17-2 Recommended storage time and uric acid). conditions for analytical samples 6. For certain analytes, the specimens/ Samples for Storage time Temperature samples should not be deep frozen. Clinical Chemistry: 1 week Refrigerator Failure to observe this can result in Immunology: 1 week Refrigerator deviating results for the following Haematology: 2 days Room temperature analytes: Coagulation: 1 day Refrigerator Fig. 17-2 Toxicology: 6 weeks Refrigerator Storage of samples Tab. 17-1 Examples of blood and urine Blood grouping: 1 week Refrigerator should enable easy constituents which should not be (at least) reidentification for stored frozen confirmatory tests Sample Analytes Serum/plasma: Lipoprotein electrophoresis Apolipoprotein A-I and B LDL-cholesterol (prevented by the addition of glycerol) Fibrin monomer positive plasma* EDTA-Blood Haematology Urine IgG Sediment Uric acid (precipitations!) *Negative test result, prolonged PTT, shortened thrombin time, shortened reptilase time (215).

7. Correct thawing A very common source of error is the inadequate mixing of deep-frozen samples after they have been thawed. Concentration gradients are produced during thawing as the concentrated solution first melts and then runs down the sides of the vessel (see Fig. 17-1).

After thawing, the sample should therefore be inverted several times, avoiding the formation of foam. Look for

41 What has to be done on specimen arrival?

Centrifugation to calculate the relative centrifugal force (rcf) is as follows: Centrifugation of clotted blood to ob- rcf = 1.118 x 10-5 x r x n2 tain serum should be performed after making sure that the blood has clotted. Where r is the mean radial distance Normally, the waiting time for blood to from the axis of rotation in centimeters clot is approximately 30 min. However, and n is the speed of rotation in revolu- patients who are on anticoagulant ther- tions per minute (rpm). 1.118 x 10-5 is apy, or those with coagulation defects a constant which is derived from the will have delayed clotting. centrifugal force as multiple of g.

Centrifugation of clotted blood to obtain serum or anticoagulated blood to Centrifugation is usually performed bet- obtain plasma is typically performed at ween 20 to 22°C. However, analytes 1000 to 1200 g for 10 to 15 min. For that are labile during centrifugation at the production of platelet-free plasma a ambient temperature, especially if the centrifugation for 15–30min at 2000 – temperature increases during centrifu- 3000 g is necessary (see Tab. 13-2). In gation, should be centrifuged at refrig- coagulation procedures, citrated whole erated temperature (4°C). However, re- blood should be centrifuged at 2000 g frigeration can lead to leakage of for 15 min (159). potassium from the cell, thus spuriously increasing its value (see Fig. 15-1). The relative centrifugal force can be Fig. 18-1 calculated either by using an empirical Specimens should not be recentrifuged Nomograph for equation or by using a nomograph as after sampling the serum or plasma. In calculation of relative shown in Fig. 18-1. The equation used doing this the ratio of plasma water to centrifugal force cell volume may be altered thus causing alterations in analyte concentra- 20.000 tions. Specimens with a gel barrier ma- 15.000 terial should never be recentrifuged. g 10.000 30.000 cm 20.000 The time and centrifugal force applied 10.000 6.000 25 speed 5.000 4.000 to the sediment of heparinized blood 20 2.000 3.000 should be such as to leave no platelets 18 16 1.000 2.000 in the plasma layer. Failure to com- 14 500 1.500 pletely sediment platelets will result in 200 12 1.000 spurious increases in potassium, lactate 10 100 9 50 dehydrogenase, acid phosphatase and 8 20 50 inorganic phosphate from platelets re- 7 10 maining in the plasma (Fig. 18-2) of the 6 3 sample (69). Fig. 18-3 shows the visual 5 20 relative revolutions control of plasma after centrifugation at 4 centrifugal per minute force various speeds. 3 Microcollection tubes with or without anticoagulants can be centrifuged to 2 obtain either serum or plasma in a mi- rotating radius crocentrifuge with an adapter that ac-

42 Samples: From the Patient to the Laboratory. W. G. Guder, S. Narayanan, H. Wisser, and B. Zawata Copyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-30981-8 Specimen processing, centrifugation, distribution

cepts such tubes. The centrifugation of Fig. 18-2 such microcollection tubes is performed Effect of platelet contamination due to at speeds ranging from 6,000 to platelet- platelet 15,000g for a minimum of 90 sec. free gradient insufficient time and plasma in plasma centrifugal force layer A preventive maintenance program for platelets applied to heparinized blood centrifuges should include a schedule platelet sediment for periodic checking of centrifugation cells cells speeds attained at a specified speed settings using a tachometer. Sufficient Insufficient time and centri- time and fugal force. Sample probe centrifugal will pick-up platelets present Sample handling after centrifugation force. in plasma giving rise to spurious results. After centrifugation, samples may be transferred directly to the analyzer. Ideally, the analyzer needle takes the To avoid contact with blood containing analytical sample by piercing the other potentially infectious materials, closed stopper after the sample has been subsampling should be avoided as far mixed. In most laboratories, however, as possible. the stopper has to be removed and the samples distributed. To prevent evapo- This is facilitated by the use of separa- ration, this should be done shortly before tors in tubes. Alternatively, distribution analysis. Subsamples should follow the can be performed by mechanical de- same rules as the primary specimens vices (Fig. 19-1, p. 44). regarding identification, storage conditions and safety aspects.

Fig. 18-3 Visual control of adequacy of centrifugation before analysis of serum

43 Continuous or batchwise?

Workflow can be defined as the steps simplified alternative to time-consuming carried out from the time a specimen and cumbersome manual aliquoting arrives in the laboratory to the time the procedures. Fig.19-1 illustrates an auto- results are reported to the physician. mated sample sorter system (127). This workflow then determines the turn- around time (TAT) of laboratory results. Consolidation of work areas such as A broader definition of workflow would routine, special chemistry and haema- encompass steps from the time the tology sections should facilitate work- physician orders a test to the time the flow. Efficient workflow is dependent results reach him. on streamlining sample processing, aliquoting and distribution steps (61). The importance of preanalytical time in terms of total TAT can be appreciated Discontinuous steps such as centrifu- from a study by Godolphin which is gation and labeling samples adversely detailed below (62). affect total turnaround time.

Tab. 19-1 Contribution of the preanalytical phase to total turnaround time (TAT) Robotics % of TAT Robots are devices that can be pro- Preanalytical 57.3% grammed to perform specific tasks. As Analytical 25.1% such, robotics is a term used to describe Postanalytical 17.6% the utilization of robots to perform specified repetitive mechanical tasks Fig. 19-1 In recent years, automated sample that are programmed and hence under Automated sample sorter systems have been introduced to electronic and computer control. sorter system cope up with the workload of large (with kind permission of P. Mountain, Auto- reference laboratories and automated Robots of varying flexibility are avail- lab, Toronto, Canada) aliquoting systems have provided a able (139). Robots with three degrees of freedom that can move about in a three-dimensional space, but are inca- pable of rotation are called Cartesian robots and have applications ranging from sampling devices on automated analyzers to pipetting stations for handling liquids (54).

Robots with four degrees of freedom are called cylindrical robots and are ca- pable of moving in and out of plane with a wrist roll. By the use of robotic arms, these cylindrical robots have been used for sample preparation tasks such as in blood typing or in multiple steps such as sample extraction, separation of aqueous and organic phases and sample injection associated with high performance liquid chromatographic procedures.

44 Preanalytical workflow and robotics

Highly flexible robots with five degrees Fig. 19-2 Robots of varying of freedom are called jointed robots degrees of flexibility and are very versatile with their wrist rotatory motion and ability to reach the Cartesian robot remote positions on an instrument. Fig.19-2 illustrates robots of varying degrees of flexibility.

Robots are also used to transport specimens to the laboratory across taped cylindrical robot tracks (118).

M. Sasaki, a pioneer in the use of robotics, used a conveyer belt system to transport samples to the robotic analyzers which in turn were connected to the conveyer belt (Fig.19-3). Sasaki has used robotic analyzers to perform a variety of tests that include jointed robot serological aggregation tests including Fig. 19-3 AIDS tests, blood transfusion tests such Conveyer belt system as ABO blood typing, cross-matching, to transport samples Rh factor testing and hormone analysis to robotic analyzers (181, 182). (with kind permission of M. Sasaki, Kochi,

Japan) In the meantime different systems of ad- ▲ vanced analytical systems including robotic regulation have been developed and incorporated into working laboratories (85, 86, 207).

While robotics can contribute to the efficiency of workflow, by their very nature they require special considerations. This is due to the fact that the movements of robotic arms are under the control of complex electronic circu- its. As such, any fluctuation in line volt- age can disrupt the operation of the robot. Hence, having access to an uninterrupted filtered power supply with back-up batteries is essential for robotic operations.

Finally, cost and space considerations have to be considered in the process of determining the suitability of robotic operations in a clinical laboratory.

45 Safety aspects during the preanalytical phase

Fig. 20-1 Steps to ensure safety are paramount to a) Disposal of needle into the protection of the health care worker. sharps container The NCCLS document GP17-T provides tentative guidelines on clinical laboratory safety (154). This document outlines steps for the maintenance and inspection of the laboratory, the general requirements for personal and laboratory safety, warning signs and labels needed, fire prevention and control, electrical and radiation safety, handling of com- pressed gases and carcinogens, chemical and microbiological hazards and hazardous waste disposal (154). a) Sampling with a safety needle

Specifically, the disposal of specimens, b) needles, tubes, and chemicals are discussed in this chapter.

Disposal of needles and other sharp objects

The disposal of all sharp objects such as needles is accomplished by placing them in leak-proof, puncture-resistant containers with appropriate labels and color coding, an example of which is illustrated in Fig. 20-1. b) Quick release holder; discard the needle into sharps container by pressing the release button. The holder then can be reset ready for reuse.

c) d)

Needle Disposal Container

FILL TO THIS LEVEL ONLY FORCING OR OVERFILLING SHARPS INTO CONTAINER MAY CAUSE SERIOUS INJURY SHARPS DISPOSAL SINGLE USE ONL Y ASTE Y SINGLE USE ONL BIOHAZARD W WARNING: This container is puncture-resistant, but not puncture-proof. To avoid injury examine the container carefully before you fill, carry, or dispose of it. For use only with VACUTAINER Brand Blood Collection Needles.

c) Single use holder: immediately after sampling, d) Container discard the needle holder combination into a sharps container.

Disposal of specimens, needles, tubes and chemicals

3 2

5 min

Immediately after sampling, activate needle safety mechanism and discard the needle into sharps container. Fig. 20-2 Safety needle for arterial collection and Reshielding of sharps and the bending Bulk body fluids such as urine, vomit, nestable sharps and breaking of needles should be feces and other body fluids may be container avoided. disposed of by flushing down the toilet.

However, should recapping become Containers of fluids such as blood bags necessary, either a one-handed scoop should be incinerated after placing in or, preferably, a reshielding device biohazard waste containers. should be used.

Simple reshielding devices are available, Chemicals which allow the phlebotomist to reshield Chemical waste can be ignitable, the needle with minimal risk of needle corrosive, reactive and toxic (152). prick injury (Fig.20-2). Examples of ignitable chemical waste Specific safety devices have also been include volatile flammable liquids such developed for the disposal of micro as organic solvents (e.g., alcohols, ace- blood collection sets (Fig. 20-3). tone, xylene, toluene, etc.), oxidizers such as peroxides and nitrate salts and flammable gases such as butane, silane Tube and sample disposal and hydrogen. These materials should be Specimen collection tubes containing labeled with the appropriate hazardous blood and which are intended for dis- chemicals label shown in Fig. 20-4. posal should be placed in biohazard bags that can withstand autoclaving. Disposal of flammable solvents via a These bags should be placed in leak- sink or flush toilet is not recommended. proof containers and then tightly closed. If such solvents are readily dissolvable in water, they could, in small amounts, be disposed of by pouring down a sink,

47 Safety aspects during the preanalytical phase

Fig. 20-3 a) followed by copious amounts of water. SAFETY LOK™ System It is best to collect flammable solvents in for reshielding needle safety cans or drums and store in a of blood collection set storage cabinet prior to collection by a disposal collector or company. Ether and chlorinated solvents should be collected in separate cans. Other solvents may be combined in one can.

Examples of corrosive solvents are strong acids such as sulfuric, hydrochloric, and phosphoric acid and bases such as ammonia (ammonium hydroxide). a) When sampling is complete apply gauze pad on puncture site. Grasp the yellow shield between your thumb and forefinger while using your remaining fin- Toxic, corrosive and inflammable gers to hold the tubing against the palm of your hand. chemicals should not be used as preservatives in the preanalytical phase. b) Never add urine to concentrated acids. Such acids should be diluted by adding them slowly down the sides of the urine container. Disposal of strong acids and corrosive materials should preferably be done by pouring down the sink, followed by copious amounts of running cold water from a tap.

Thus when urine is collected in hydrochloric acid, the first portion should be collected before acid is added. b) With the tubing held taut, advance your thumb and forefinger to slide the safety shield forward until an audible “click” is heard. Toxic chemical waste such as toxic metals can pose a threat to ground water. c)

c) The click confirms that the shield is locked into place, covering the needle. Dispose the blood collection set in a suitable container.

48 Disposal of specimens, needles, tubes and chemicals

Fig. 20-4 The Environmental Protection Agency in Safety labels of the United States of America (EPA) lists United States and chemicals that constitute toxic waste DANGER European origin (222). A European list of maximal allow- able concentrations of chemicals in air, HAZARDOUS WASTE water and foodstuffs is also available (42). STORAGE AREA Finally, to be knowledgeable about chemical hazards, each laboratory should have a file of material safety data DANGER sheets (MSDS) that list the hazardous properties of each chemical, and which FLAMMABLE can be readily consulted in cases of emergency or when there is doubt as to PELIGRO their hazardous nature. INFLAMMABLE

CORROSIVE

irritant corrosive harmful to the environment

explosive oxidizing flammable toxic

49 What is needed before blood transfusion?

Ensuring patient and sample identity ● For cold agglutinin determination, the While processing blood and blood blood sample must be kept at 37°C un- products 41 % of the reported defects til serum has separated. were shown to be related to the prean- ● Citrate blood is necessary if identifi- alytical phase, 55 % to the postanalyti- cation of erythrocyte-bound antibodies cal phase and only 4 % to the analyti- requires elution. cal phase according to a study of ● EDTA blood samples are used for the Boone et al. (20). The group at highest detection of in-vivo complement bind- risk are patients issuing 10 or more ing by erythrocytes. For special erythro- units per day. cyte antigen testing, citrate-anticoagulated blood should be used. Most haemolytic transfusion reactions ● Samples of blood contaminated with result from discrepancies in patient or Wharton‘s jelly may agglutinate spon- sample identity. The frequency of ad- taneously. The cells should be separated ministration of a wrong transfusion by by washing three times with 0.9% so- means of mismatching the samples is dium chloride solution. 1:60000 transfusions. Therefore, request ● Interferences of the agglutination forms for blood transfusion must con- reaction involve either pseudo- or tain the first name, last name, the date polyagglutination. Pseudoagglutination of birth and, if possible, an identification (rouleaux) can be caused endogenous- number unique to the patient. ly or exogeneously (Fig. 21-1). Before blood sampling is performed, ● Exogenous factors are infusions of active control of patient identity has to dextran, polyvinyl-pyrolidon or fibrino- be thoroughly ascertained by asking gen; among endogenous variables are the subject for his or her last name, first disproteinemias in immunocytoma, liver name and date of birth. Blood samples cirrhosis or hyperfibrinogenaemia. must be collected in correctly labeled ● Polyagglutination is a condition in tubes. The name of the person who has which red blood cells are agglutinated drawn the sample must also be docu- by a high proportion of group-compati- mented. ble sera. The same phenomenon occurring in vitro is called polyagglutination The right sample (28). This can occur during viraemia or bacteriaemia (endogenously) or after ● Serum is used for immuno-haemato- exogenous bacterial contamination of logical tests. Anticoagulants such as patient erythrocytes. Bacterial enzymes EDTA or citrate prevent complement modifiying erythrocyte antigens releas- activation. Consequently, complement- ed are one of the causes of in vitro and activating antibodies are not detectable in vivo polyagglutination. T-polyaggluti- in such plasma samples. nation is caused by a microbial neu- ● The use of haemolytic samples is raminidase, the TK-polyagglutination normally not allowed because antibody- by a β-galactosidase or the acquired B- induced haemolysis can be masked. antigen by deacetylase. ● The patient sample for pretransfusion Non-microbial polyagglutinations are testing should not be taken less than 72 also known. These are caused by somatic hours prior to the test. mutation of a pluripotent haemopoetic ● Each patient sample for immuno- stem cell (Tn) or congenital genetic haematological examination must be defects. stored at 4–6°C for one week following the test. 50 Samples: From the Patient to the Laboratory. W. G. Guder, S. Narayanan, H. Wisser, and B. Zawata Copyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-30981-8 Special aspects in immune haematology

● All immuno-haematological results should be observed and interpreted by two independent persons who should sign for the correctness of the procedure.

Storage of blood for transfusion Blood units have to be stored in special refrigerators equipped with an automatic temperature recording system and an optical or audible alarm system. The low alarm activation should be set at 3.5 ± 0.5°C and the high alarm activation at 5.5 ± 0.5°C. The surface Fig. 21-1 of the blood container must be clean Pseudoagglutination and dry. Any item that could result in (rouleaux) the puncture of blood containers should be removed from the storage area. Fig. 21-2 Products for transfusion must be stored Blood typing by gel filtration test separately from reagents or used pilot tubes. The storage refrigerator should be kept clean. A standard procedure for cleaning the storage refrigerator is mandatory (5).

What has to be done before transfusion? Both on delivery of the blood unit and prior to transfusion, all information on the container and the accompanying transfusion form have to be checked for identity. The ABO-status of the donor must be evaluated immediately before transfusion by means of a bedside test. In the case of autologous transfusion, ABO status has to be checked for both the blood unit and the recipient by bedside testing. The residual blood of the transfused unit has to be kept for at least 24 hours after infusion at 2–8°C (230).

51 Why a separate tube for the coagulation test?

If several evacuated Specimen collection amount of headspace above the blood blood collection tubes The results of coagulation screening is also a variable that can affect the are used, the specimen tests such as the prothrombin time (PT, APTT result (2, 194). Apparently, the infor coagulation testing should be collected in Quick Test) and the activated partial crease in surface area in a tube fa- the second or third tube thromboplastin time (APTT) are decisive- vours platelet activation with the subse- in order to minimise ly influenced both by the anticoagulant quent release of platelet factor 4, contamination with used, its concentration and ratio to which in turn neutralises some of the tissue thromboplastin. blood, and by the manner of collection heparin causing a shortening of the and further processing of the specimen. APTT value (2, 194). Citrate can be regarded as standard anticoagulant (105). The recommended citrate concentration is 0.105 mol/L (3.2 %) (89, 155). A citrate solution buffered to pH 5.5 is Concentration of the anticoagulant preferable to an unbuffered solution as Prothrombin time (PT) reported as the in the buffered solution the pH of the international normalised ratio (INR) is specimen is closer to the physiological sensitive to the concentration of citrate. range (146). If the haematocrit is INR values, especially with a respon- above 0.55 a correction is necessary Strict observation of a sive PT reagent similar to the WHO even if the ratio of anticoagulant to ratio of 1+9 (citrate thromboplastin, with an international blood is 1+9 (1:10). This is because the solution to blood) for sensitivity index (ISI) equal to 1, are plasma compartment is reduced, result- determination of the generally higher with a 0.129 mol/L ing in an excess of citrate in the speci- APTT and other coagu- (3.8 %) than with a 0.109 mol/L (3.2 %) men container which in turn complexes lation screening tests is sodium citrate (2, 142). The differences with the calcium ions added during recommended. in INR between the two concentrations measurement of the PT and APTT. Thus of citrate can vary from 0.7 to 2.7 INR the clotting time is prolonged. units (2).

Hence a laboratory should not inter- Specimen processing change the two concentrations of cit- After carefully inverting the specimen rate when PT is reported in INR units on container several times to exclude clot patients who are undergoing oral anti- formation and checking that the ratio of coagulant therapy. anticoagulant to blood is correct, the closed specimen tube is centrifuged at In addition to maintaining the nominal 2000 g for 10 minutes (155). Specimens anticoagulant to blood ratio (1+9), the with invisible clotting or haemolysis should not be used as activation of clot- Fig. 22-1 ting factors may have taken place. In Effect of haematocrit 80 addition, it is very important to look out on the APTT using a for lipaemic or icteric specimens as buffered citrate solution 60 (0.129 mol/L) these can cause interference in the

▼ 40 case of photo-optical measurements. APTT (sec)

20 Unopened specimens for the performance of coagulation screening tests (PT and APTT) should be transported 0.3 0.6 0.9 to the laboratory at room temperature haematocrit (fraction) (22–24°C) but not on ice.

52 Special aspects in immune haematology

The table in the Annex gives details on within two weeks. In specimens frozen For single factor assays, specimen stability for the different coat –70°C the factor VIII activity is stable particularly of factor agulation tests. for up to one year. VIII, the blood should be cryocentrifuged (+4°C) The following general principles can as soon as possible Evaluation of fibrinolysis and serve as a guide. after collection and the ● If the test is to be performed immedi- monitoring of fibrinolytic therapy plasma frozen at –20°C ately the specimen is kept at room tem- After a freezing and thawing process, to –70°C. perature. The plasma can be left standing identical results are only obtained if the on top of the packed cells after cen- plasma contained no fibrin monomers, trifugation. fibrin degradation products or heparin ● The collection vessel should be closed before freezing. to avoid changes in pH due to evapo- In plasmas which contain fibrin mono- ration of volatile carbonic acids. mers gelling processes after thawing ● Exposure to high temperatures (in- can result in negative fibrin monomer cluding direct sunlight) must be avoid- detection, prolongation of the APTT and ed at all costs. prolonged thrombin and reptilase times. The measurements of ● Specimens should not be refrigerated In plasmas with considerably prolonged the PT and APTT are (+2 to 8°C) as cold activation of factor thrombin times (heparin, FDP) instability least influenced if the VII, and also of factors XI and XII, can after thawing can lead to shortening or plasma is analysed at lead to shortened clotting times in the even normalisation of the thrombin time room temperature within respective screening tests. and APTT after thawing (216). 2 hours. ● For tests to be performed at a later Specimens for monitoring fibrinolytic date platelet-free (< 5000/µL) citrated therapy with streptokinase or uroki- plasma is aliquoted and frozen in nase, for example, should be collected closed tubes. into tubes containing a mixture of EDTA ● Frozen specimens should be thawed and aprotinin as this combination im- quickly in a water bath at +37°C and mediately inhibits the streptokinase or mixed thoroughly, making sure that any urokinase induced plasmin activation cryoprecipitates are completely dis- (146). For this, aprotinin in a concen- solved. Repeated freezing and thawing tration of 150 kIU/mL blood should be is not recommendable. combined with trisodium citrate (10 mmol/L) or EDTA (4.2 mmol/L). Specimen storage Unlike FDP measurement, which requires a tube containing 10 units of The plasma should be left standing on thrombin and 1835 units of soybean the sedimented cells and used within 2 trypsin inhibitor/mL of blood detection, to 6 hours (for details see Annex). If the D-dimer, which reflects the fibrinolytic storage period is to exceed 4 hours the activity, can be performed in citrated plasma specimens can be kept in the plasma. refrigerator for 4 weeks at –20°C, and with rapid freezing to –70°C the stor- The activity of rt-PA (recombinant tissue age time can be extended to 6 months plasminogen activator) in the blood (83, 155). can be effectively inhibited with a mixture of 5 mmol/L D-phenylalanine-pro- Factors V and VIII are unstable. The fac- line-arginine-chloromethylketone and tor VIII activity (VIII.C) of the samples 10 mmol/L citrate or 4.2 mmol/L EDTA frozen at –20°C should be measured (138, 142).

53 Blood cells are sensitive!

Optimum anticoagulant cytes being relatively more stable and The International Council for Standard- neutrophils and monocytes more likely ization in Haematology (ICSH) has rec- to be influenced when stored in EDTA,

ommended dipotassium EDTA (K2-EDTA) introduces a variable that is com- (ethylenediaminetetraacetic acid) as pounded by the variation in the cluster the anticoagulant of choice for the col- analysis software packages used by lection of blood specimens intended for the different analyzers. blood cell counting and sizing (170). Ratio of anticoagulant to blood K2-EDTA was selected in preference to Na2-EDTA because of the greater solu- The volume of blood drawn should ensure bility of the potassium salt compared to maintenance of the recommended the sodium salt. concentration range for the EDTA salt of 1.2–2.0 mg/mL (180). With all EDTA salts, the cells shrink, thus affecting the centrifuged but not the cal- Since the concentration of EDTA has an culated haematocrit. Because of the effect on neutrophil morphology, the

lower pH of Na2- and K2-EDTA com- quality of the peripheral blood smear pared to K3-EDTA, the cells swell, thus can be compromised if attention is not compensating for osmotically induced paid to the anticoagulant to blood ratio cell shrinkage. Calibration of electronic and the time elapsed between blood blood cell counters for mean corpuscu- collection and preparation of the smear. lar volume (MCV) using the microhaematocrit value obtained from blood At concentrations of EDTA of up to 1.50

specimens collected in either Na2- or K2- g/L of blood in blood specimens not EDTA have been reported to give ac- over 1 hour old, minor changes appear ceptable results, in contrast to the unac- in neutrophil morphology. As the con- ceptable results obtained when micro- centration of EDTA increases, more seri- haematocrit values obtained from blood ous changes such as loss of bridges be-

specimens collected in K3-EDTA were tween lobules, loss of cytoplasmic used to assay commercially available boundary and early cross-over occurs control material (170, 188). within the first hour.

With a variety of techniques now incor- The effect of increase in the concentration porated in the new automated instru- of EDTA over the nominal value decreases ments for counting, sizing and provid- the centrifuged microhaematocrit value, ing a 5-part white blood cell differential the decrease being more pronounced

count, the question has been asked with K3-EDTA than with K2-EDTA. whether any salt of EDTA is the adequate anticoagulant for haematology (169). However, with automated instruments, the mean corpuscular volume was not

Platelets change from their discoid to a influenced at K3-EDTA concentrations spherical shape upon blood collection up to 10 times the nominal value; and

in EDTA, thus introducing an error in the results obtained with K2-EDTA were mean platelet volume (MPV) determina- shown to be dependent on the instru- tion. ment used (63). The influence of EDTA on the stability of white blood cell populations, lympho-

54 Special aspects in haematological analysis

Using the recommended concentration of EDTA salts (K2- and K3-EDTA) and performing analysis between 1 and 4 hours after blood collection, no significant difference was seen in results obtained with blood collected in either of the 2 anticoagulants (63).

Specimen collection and handling Thorough mixing of the blood specimen with the anticoagulant by inverting the tube several times is a prerequisite. The type of mixer (rocking versus rotary) used may affect the extent of mixing, pressing the platelet count. The problem Fig. 23-1 especially if the tube is overfilled, thus can be recognized by examination of Satellism of platelets yielding inaccurate results (166). the peripheral blood smear, and also on granulocytes by being alerted by the platelet flags (neutrophils) or alarms in the instrument (39). If a blood collection Transport, storage and Accurate platelet counts on subjects tube is drawn to stability of analytes showing EDTA-induced platelet satel- one-half of its nominal Because of the variations between lism can be obtained by diluting blood volume, the effective newer automated instruments, including from a finger prick or by collecting concentration of EDTA reagents, it has been recommended blood with citrate as the anticoagulant. would be unsuitable for preparation of a that the EDTA anticoagulated blood be peripheral blood smear analyzed within 6 hours of collection intended for white (150, 170). In some cases, however, Special considerations for platelet content measurements blood cell differential this time is already too long to ensure count (180). constant results. Only haemoglobin and To assess in-vivo activation of platelets platelet number are stable over this by measurement of constituents in the Blood collection tubes time. Likewise, stability at refrigerator platelet α-granules such as platelet must have an air space temperature is analyzer-dependent (for factor IV (PF4), β-thromboglobulin, representing at least details see Annex). For the reasons giv- fibronectin and platelet-derived growth 20% of the volume of en above, the blood smear should be factor, activation of platelets after blood the tube to facilitate prepared within 1–2 hours of blood collection should be minimized. This mixing (170). collection. Extended storage for up to can be accomplished either by prevent-

24 hours is not recommended. ing the formation of thromboxane-A2 or by maintaining high levels of cyclic AMP within the platelets. An additive Pseudothrombocytopenia mixture used for this purpose consists of Platelet clumping or agglutination, and 0.11 mol/L citric acid, 15 mmol/L theo- platelets adhering to neutrophils (platelet phylline, 3.7 mmol/L adenosine and satellism (Fig. 23-1)) have sometimes 0.198 mmol/L dipyridamole with the fi- been observed with EDTA anticoagu- nal pH being adjusted to 5.0 (38, 138). lated blood, such changes becoming PF4 levels in blood collected in this ad- progressive with the time elapsed after ditive mixture called CTAD is almost 10 collection. This phenomenon elevates times less than that obtained in a con- the white blood cell count while de- ventional citrate tube (223).

55 Everything from a drop of blood?

In clinical chemistry, a number of re- cause the amount of plasma available agent- and analyzer-specific problems to the reactive zone of the test strip have to be considered. Thus many varies at different packed cell volumes. interference factors act in a reagent- specific way. In addition there are Dry chemistry methodologies on the differences between analyzers and one hand offer the possibility of sepa- analytical principles (i.e. ”dry“ and ”wet“ rating the analyte from many disturbing chemistry, direct and indirect potentio- components in the matrix. On the other metry). In this chapter, a few examples hand the matrix is more diluted in wet only are demonstrated (66, 213, 239). chemistry procedures, resulting in lower analyte and possible interfering concentrations. Special aspects when using so-called “dry chemistry“ (99, 202) When a carrier-bound reagent analyzer Different results using methods with for capillary blood is used, the correct and without deproteinization? sampling of capillary blood samples is Protein molecules in serum-/plasma of decisive importance for the reliability occupy a defined volume, dependent of the results. The producer of the on the concentration and the size of the Reflotron® System (Roche Diagnostics, protein. As a result, the concentration Germany) recommends: of low molecular solutes (e.g. glucose, electrolytes etc.) in protein-free filtrates ”When a large free-hanging droplet are found to be approximately 5% has formed, it should be applied to the higher than in untreated serum-/plasma sample application field of the test car- samples without deproteinization. The rier without directly touching the carrier influence of deproteinization on the with the finger. For an additional determination of low molecular weight measurement it is necessary to prick the substances which are dissolved in finger at a different site.“ serum-/plasma water, is shown in Fig. Results in a whole blood analyzer may 24-1 (25). The volume displacement depend on sample haematocrit, be- effect of proteins is especially important for the determination of electrolytes us- Fig. 24-1 ing direct potentiometric in comparison Change of concentra- to indirect measuring procedures (88) tion of low molecular as well as for determination in depro- weight substances after deproteinization teinised whole blood (e.g. glucose). (from (25)) The volume displacement effect of lipids A similar displacement effect is observed with triglycerides in blood. At 5000 mg/dL (57 mmol/L) triglyceride concentration, direct potentiometry gives about 5% higher (and more true) results of electrolytes compared to flame pho- low molecular volume of tometry or indirect (after dilution) poten- weight substance protein tiometry (110).

56 Special aspects in clinical chemistry

Whole blood versus plasma glucose plasma/serum When comparing glucose in whole blood with that in plasma, similar, but CO2 larger differences are observed. Since blood cells have a higher protein and HCO + 3 K lipid content and glucose is not equally cell glucose + distributed between the intracellular Na and the extracellular space, results obtained in plasma are approximately CI 15% higher compared to whole blood, when related to the same volume. The The triglyceride concentration in the Fig. 24-2 WHO (3) and the ADA criteria (6) for sample falls while that of free glycerol Electrolyte fluxes the diagnosis of diabetes mellitus are rises. The extent of this effect varies from between blood cells and plasma/serum therefore different for plasma and person to person and does not correlate during storage of whole blood. with the initial triglyceride concentra- whole blood tion. In addition, the composition of + + -- -- plasma lipids determines their behavior Electrolytes (Na , K , Cl , HCO3 ) during centrifugation. Chylomicrons and If during transport and storage of whole their remnants tend to float to the top blood the glucose concentration falls layer of plasma, whereas other lipopro- below a critical concentration, the cells teins remain equally distributed. This has lose their intracellular potassium and to be considered when primary tubes take up sodium in its place. If CO2 are used in analyzers (131). escapes from a blood sample, the cells lose bicarbonate and replace it with Creatinine chloride from the surrounding plasma (chloride shift) (Fig.24-2). This limits the The concentration of non-creatinine stability of whole blood for the plasma/- chromogens increases at room tempera- serum analysis of electrolytes. Interes- ture, this effect being more pronounced tingly, the increase in plasma potassi- in whole blood than in serum/plasma um is higher in refrigerated blood (the higher the temperature, the greater samples compared to samples stored at the effect). This increase – which varies room temperature (84). This is caused from person to person and is independ- by an inhibition of the cellular Na+,K+- ent of the initial creatinine concentra- ATPase activity brought about by cold. tion – occurs when creatinine is determined by the Jaffé method (145). Trace elements Recommendation: Contamination plays a significant role in trace element analysis. Blood should In clinical chemical analysis, method- therefore be obtained in a suitable and analyzer-specific prenalytical influ- sampling system that has been declared ences and interferences have to be tak- trace element-free by the laboratory. en into consideration. Results obtained with one system are not transferable to Lipids others without experimental proof.

Storage alters triglyceride concentration Detailed information may be obtained due to the action of endogenous lipases. from reagent and analyzer producers.

57 Special tubes for hormones?

Sampling, storage and transport decrease both insulin and proinsulin for analysis by immunochemical values. methods Processing of blood promptly upon Sensitive immunochemical methods clotting and freezing of serum at –70°C lend themselves to the measurement of provides long-term stability for analytes trace quantities of labile hormones, such as gastrin, pepsinogen-1, and proteins and other analytes in blood. insulin. Because of the wide range of analytes measured by immunoassays, this chap- Collection of blood in an ter will attempt to focus on a few repre- appropriate anticoagulant sentative analytes (12). Most analytes determined by immunochemical methods can be measured in Posture and timing serum and/or heparinized plasma. Variables such as posture during blood sampling and diurnal changes have to Collection of blood in EDTA and be taken into consideration. promptly freezing the plasma has been found to be adequate for preserving Postural variations have a significant labile polypeptide hormones such as impact on renin, elevation of enzyme endorphin, vasoactive intestinal peptide, activity being observed when moving substance P and pancreatic peptide. from the recumbent to the erect position. By its inhibitory effect on metallopro- Cortisol has a peak value between 4 teinases EDTA plasma is also recom- a.m. and 6 a.m (see Fig. 5-2). mended for ACTH, parathyroid hormone and glucagon. Hormones such as growth hormone, lutropin (LH) and follitropin (FSH) are released in bursts, and as such, several Collection of blood with proteolytic blood specimens taken within closely enzyme inhibitor timed intervals are needed to establish A proteinase inhibitor called aprotinin a median value. (also known by its trade name Trasylol) added to an anticoagulant such as ED- TA or heparin has found application in Refrigeration and freezing the stabilization of labile polypeptide Some hormones such as insulin, proin- hormones and enzymes (137). Since sulin and C-peptide can be stabilized aprotinin inhibits kallikrein, its potency by merely placing blood specimen con- is expressed in terms of kallikrein in- tainers on ice immediately after collec- hibitory units (KIU). The concentration tion. Such specimens should be promptly of aprotinin used for the preservation centrifuged, preferably in a refrigerated of labile hormones and enzymes centrifuge at 4°C, and the serum kept ranges from 500 to 2000 KIU/mL. frozen until assayed. Of course, the frozen Thus, a mixture of EDTA aprotinin has specimen should be completely thawed been used to stabilize glucagon, prior to assay. It is also important that ACTH, renin and certain gastrointesti- haemolysis be avoided, since this will nal hormones such as β-endorphin, secretin, neurotensin, gut glucagon, so-

58 Preanalytical factors in immunoassays matostatin and vasoactive intestinal hibitor such as nafamostat mesylate is peptide (137). added to EDTA, the stability of comple-

ment components (C3a, C4a and C5a) is In one study it was demonstrated that significantly improved. Furthermore, glucagon levels measured by RIA in while the activity of complement com- EDTA plasma were approximately 26% ponents doubles with each freeze-thaw higher than in plasma obtained from cycle when blood is collected with blood collected in a mixture of 1.5 mg EDTA alone, no such discrepancy is EDTA and 2000 KIU of aprotinin per mL seen in samples collected in EDTA sup- of blood (Tab. 25-1 ). The high gluca- plemented with nafamostat mesylate gon level in samples collected without (227). aprotinin was due to the fragments of hormone produced by proteolytic en- Traditional anticoagulants such as EDTA zyme degradation being apparently and heparin have been ineffective in recognized as an intact molecule by stabilizing a labile constituent such as the antibody used in the assay. In addi- parathyroid hormone related protein tion, some of the radio-labeled hor- (PTH-RP). This tumor marker is so unsta- mone underwent proteolytic enzyme ble that less than 10% of the original degradation, thus limiting the amount activity remains after 16 hours of stor- of radio-label available to compete age at room temperature in blood spec- with the hormone in plasma for binding imens collected in either heparin or sites on the antibody (48). EDTA (164). The optimum mixture for blood collection is EDTA (1.5mg/mL of Tab. 25-1 Effect of aprotinin on glucagon blood), aprotinin (500 KIU/mL), leu- measurements in EDTA plasma peptin (2.5mg) and pepstatin (2.5 mg). With this additive mixture, PTH-RP is sta- EDTA + Aprotinin EDTA ble for up to 1 hour at room tempera- n2121ture and for 24 hours if maintained re- Mean pg/mL 386 518 frigerated at 4°C (164). % Difference –25.5 For the measurement of catecholamines A mixture of lithium heparin and in plasma, blood should be collected in aprotinin has also been used to stabi- an anticoagulant mixture such as EGTA lize immunoreactive somatostatin, se- (90mg/mL) supplemented with sodium cretin, glucagon, C-peptide and metabisulfite or glutathione (60mg/mL). cholecystokinin-pancreozymin. Even with this additive mixture, the blood has to be kept cold in an ice bath and centrifuged in a refrigerated Collection of blood in an centrifuge. The plasma should then be anticoagulant-additive mixture transferred to a plastic vial, capped and stored in a freezer at a temperature There are instances where EDTA alone below –20°C until ready for analysis. is not sufficient for stabilizing an ana- Frozen plasma prepared under the lyte. Thus, even when blood is collected above conditions preserves catechol- in EDTA and the plasma is separated amines for at least 3 months (19). promptly and stored under ideal conditions, there is activation of complement. However, when a synthetic protease in-

59 Blood cells can provide important information

Separation of cells from peripheral In the procedure, diluted whole blood is blood for cellular analysis. layered over the FICOLL-HYPAQUE mixture and centrifuged at room tempera- For the collection and transport of pa- ture (20 °C) for 40 min. The centrifugal tient blood samples for cell analysis force attained at the interface is 400 g. propylen tubes are required, because Since the FICOLL-HYPAQUE medium is A false negative result cells are adsorbed on glass and poly- less dense than red blood cells and in HLA typing will have serious consequences ethylen (178). granulocytes but more dense than mono- when a patient or organ Separation of a pure population of cells nuclear cells (lymphocytes and mono- transplant donor is be- such as lymphocytes from peripheral cytes) and platelets, the mononuclear ing typed for HLA com- blood are required for tests such as cells will remain at the plasma-Ficoll- patibility. human leukocyte antigen (HLA) typing Hypaque interface. Subsequent washes studies. If the cell preparation is signi- eliminate platelets from the lymphocyte ficantly contaminated with granulocytes pellet. and platelets, they may bind some of A recent study described the use of the HLA-antibody thus yielding a false either heparin or buffered citrate as an negative result. anticoagulant, polyester gel barrier and liquid density gradient medium in How to purify lymphocytes an evacuated tube for blood collection. A 20 min centrifugation step at 1,500 g Lymphocytes can be purified using the at ambient temperature resulted in iso- FICOLL-HYPAQUE centrifugation proce- lation of peripheral blood mononuclear dure. cells (PBMC) over the gel barrier and A typical FICOLL-HYPAQUE mixture separation from erythrocytes and gra- consists of 10 parts of 33.9% Hypaque nulocytes trapped underneath the gel with a density of 1.2 kg/L and 24 parts (132, Fig. 26-1). of 9% aqueous solution of Ficoll. Both Hypaque and Ficoll solutions should be Effect of anticoagulants on the mixed at room temperature. The final recovery of granulocytes Ficoll concentration in the mixture should be 6.4% and the density of the Granulocytes can be recovered from mixture 1.077 kg/L (23). FICOLL-HYPAQUE centrifugation by re-

Fig. 26-1 Processing steps with AFTER BLOOD CENTRIFUGE AFTER FOR COLLECTION CENTRIFUGATION TRANSPORTATION the cell separation Gently invert 8 times. At room temperature Gently invert once. tube For 20 minutes Acceptable In a horizontal rotor blood (swing-out head – volume appropriate tube adapters) At 1500 g – 8mL – 7mL For best results, centri- – 6mL fuge within two hours 1 of blood collection. 2 3 4 anticoagulated plasma cell whole suspension blood lymphocytes (plasma and and monocytes density mononuclear gel cells) barrier gel gradient barrier fluid density gradient erythrocytes fluid and neutrophils

60 Special aspects in cellular analysis moving the fraction above the FICOLL- cations have, because of their rapidity, HYPAQUE layer. 0.4 mL of 4.5% dextran supplanted the time-consuming FICOLL- and 1 mL of heparinized plasma are HYPAQUE cell separation procedure then added to this fraction. The residual (102). Even the lysis techniques have pro- red blood cells are allowed to sediment gressed from the original hypotonic at 4°C for 40 min. Dextran promotes lysis techniques to the currently commer- red blood cell rouleaux formation and cially available non-hypotonic methods. favors sedimentation. The supernatant In a study performed to evaluate the is thus enriched with granulocytes. effects of various anticoagulants on both Boyum found that EDTA gave better lysis methods and the FICOLL-HYPAQUE granulocyte yields (average 59%, range procedure, it was demonstrated that if 43–71%) than heparin (average 49% analysis is performed on the same day, range 38–61%) (23). EDTA, ACD or heparin gave equivalent results. However, beyond 24 hours there is a significant decrease in granulocyte Relative merits of anticoagulants viability in EDTA. Furthermore, K3EDTA and nutrients for cell separation has been reported to be associated and stability with a loss of function in lymphocyte ACD (acid citrate dextrose), heparin mitogen stimulation assays. Granulo- and EDTA have all been used for the cyte viability was best for heparin. Lym- separation of mononuclear cells. How- phocytes were preserved equally in he- ever, regardless of whether EDTA, ACD parin and ACD. Although ACD could or heparin was used as anticoagulant, be used as an alternative to heparin, it has been reported that the lympho- there is a tendency for platelets to ag- cyte fraction was contaminated with gregate in ACD, especially if specimens granulocytes if the blood sample was cannot be analyzed promptly (34). over 14 hours old. Red cell contamina- If a flow cytometric test of the leuko- tion is a problem with 2-day-old EDTA cytes is not performed during six hours blood (161). after sample collection, the cell number From other authors it is reported that Fi- should be determined immediately af- coll gradient preparations of mononu- ter sampling the EDTA blood. That is al- clear leukocytes from EDTA-blood sam- so necessary, if the immunophenotyp- ples are contaminated by neurophile ing is performed with heparin blood leukocytes and erythrocytes (178). (50 IE heparin/mL sample) (178). On Nutrient dilution media would appear the other hand, EDTA blood has the ad- to influence the quality of FICOLL-HY- vantage that the loss of mature cells of PAQUE lymphocyte separations. Thus, the myeloid lineage by adsorption on blood collected in heparin and supple- the tube wall and platelet aggregation mented with glutamine and gentamicin is diminished (178). For the platelet gave good FICOLL-HYPAQUE separa- function analysis by flow cytometry tions with blood stored at room temper- technique the use of citrate blood is re- ature for up to 3 days (135). commended despite some disadvantages. EDTA and heparin should be Whole blood lysis for flow avoided because of their effects on cytometry applications glycoprotein structure and artifactual platelet activation (185). Whole blood lysis techniques for flow cytometric immunophenotyping appli-

61 How to handle genes

Restriction fragment length reported to retard or even completely polymorphism (RFLP) inhibit the amplification of DNA during To help understand some of the preana- the polymerase chain reaction (PCR) lytical problems, let us visualize a (87). However, treatment of heparinized person whose DNA is being examined blood with heparinase to cleave heparin for RFLP to detect some genetic defect or the separation of leukocytes by or gene rearrangements. When DNA is centrifugation followed by at least two cleaved using restriction enzymes, frag- washings with a saline buffer is reported ments of varying sizes result depending to overcome the effect of heparin (16). on whether or not the polymorphism is within the restriction enzyme cleavage Actually, other anticoagulants such as site (140). EDTA and ACD also inhibit restriction enzymes. However, standard ethanol It has been reported that aberrant or DNA precipitation techniques remove unexpected restriction fragments were both EDTA and ACD while heparin is obtained on DNA being examined for not removed (36). T and B cell gene rearrangements using blood collected in heparin (88). Heparin should not be used as anti- Such aberrant fragments were not coagulant in the molecular biological found upon restriction enzyme digestion analysis of blood. of DNA obtained from blood collected If heparin is present in sample to be in either EDTA or ACD. Since these analysed, it should be eliminated be- aberrant fragments found using hepa- fore application of the sensitive rtPCR. rinized blood may be confused with This can be done by application of he- gene rearrangements, the choice of parinase or precipitation of mRNA with anticoagulant for blood collection LiCl (94, 95). intended for certain molecular biology procedures becomes critical (214). Erythrocytes can likewise inhibit taq DNA polymerase by formation of haematin (140). Polymerase chain reaction (PCR) Heparin at a concentration as low as Red blood cells can be removed by 0.05 U/reaction mixture has been selective lysis with a buffer mixture consisting of 155 mmol/L ammonium Fig. 27-1 chloride, 10 mmol/L potassium bicarbon- Effect of anti- sample collected sample collection in EDTA or ACD in heparin ate and 0.1 mmol/L EDTA adjusted to coagulants on RFLPs extract DNA pH 4.

Alternatively, the cytoplasmic membrane of all cells can be dissolved with a incubate with restriction buffer mixture containing the non-ionic endonuclease detergent Triton-X100 leaving behind examine restriction fragments by electrophoresis the nuclei of white blood cells from

band 1 band 1 which DNA can be extracted. How- band 2 ever, this technique will result in the loss heparin inhibitory of extranuclear DNA and RNA to the effect results in incomplete formation of RFLPs. supernatant; hence, mitochondrial DNA will not be able to be extracted (27).

62 Special aspects in molecular biology

Considerations for the isolation of Generally, non-ionic detergents such as DNA and RNA Tween 20 and Triton X-100 do not inhibit Taq polymerase in concentra- Classical techniques are based on tions of less than 5% (v/v). lysing cells with lysozyme, alkali or detergents. The removal of proteins However, ionic detergents such as and other contaminants is effected by sodium dodecylsulfate (SDS) which are incubation with protease and/or extrac- generally used in concentrations up to tion with phenol or chloroform. as high as 2.0% (w/v) can be inhibitory to Taq polymerase, since a concen- The extract should be concentrated by tration greater than 0.01% (w/v) has precipitation with ethanol in the pre- been found to be inhibitory. sence of sodium or ammonium acetate. If necessary, RNA can be removed us- Other ionic detergents such as sarkosyl ing DNase-free RNAase. and sodium desoxycholate have been shown to inhibit Taq polymerase at The advantage of using proteinase K is concentrations greater than 0.02% that, in addition to releasing DNA from (w/v) and 0.06% (w/v) respectively. chromatin, it also destroys nucleases Hence, it is important that ionic de- which would otherwise reduce the tergents be efficiently removed by average molecular weight of DNA phenol/chloroform extraction and by (140). However, proteinase K has to be ethanol precipitation and subsequent removed before the isolated DNA can washing of the DNA pellet. be subjected to restriction enzyme treatment. Also, Taq polymerase can be Even with non-ionic detergents such as degraded by proteases. nonidet P40 (NP40), where 1% (v/v) has no effect on reverse transcriptase Proteinase K can also be inactivated by enzyme, 0.1% (v/v) can inhibit Taq heating the cell lysate or purified DNA polymerase. to 95°C for 10 min. Hence, it is important to perform pre- Residual phenol can inhibit Taq poly- liminary experiments to establish the merase. Hence, a final extraction with effective concentrations of detergents chloroform isoamylalcohol (49:1v/v) and other known inhibitory reagents should be performed after phenoliza- that may affect DNA amplification by tion to remove any trace quantities of PCR. phenol remaining in the aqueous phase. Chaotropic agents such as guanidinium isothiocyanate have been fre- Salts used to subsequently precipitate quently used for the extraction of DNA DNA should be removed by washing or RNA. the pelleted DNA with 80% ethanol. The advantage of using 5 mol/L The type of detergent used for cell lysis guanidinium isothiocyanate for RNA may influence DNA amplification by isolation is that it is able not only to PCR. remove proteins from RNA but also to denature ribonucleases which would otherwise degrade RNA (140).

63 How to handle genes

Stabilizing RNA during transport, ing of native samples to 2 hours (140). storage and sample preparation By using 5 mol/L guanidinium isothiocyanate, the sample is stabilized by de- A number of factors influence RNA naturation of RNAses for approximate- stability. This rapid decrease in reverse ly one week at room temperature. transscriptase PCR sensitivity has been shown to be due to RNAses present in In terms of isolation of undegraded the sample, limiting the time of process- RNA, one must eliminate ribonuclease (RNase) contamination. These enzymes Fig. 27-2 are so stable over a wide range of pH Use of anticoagulants a and resistant even to boiling that and stabilizers in glassware, reagents and even the in- sampling of blood for vestigator‘s fingers are a source of gene analysis potential contamination. Glassware H should be treated with a 1% solution of E RFLP P in and diethylpyrocarbonate (DEPC) which is A PCR! known to inhibit RNases. Residual R I DEPC, however, should be thoroughly N removed by autoclaving the glassware in order to convert DEPC to carbon dioxide and water and subsequent heat-treatment of the glassware at b 250°C for 4 hours. Even sterile disposable plastic ware is not always free of RNAse! In case of contamination the materials like pipette EDTA RFLP tips and tubes should be autoclaved in Citrate in and hot air before use. or PCR ! ACD Contamination control

DNA from exogenous sources such as a person’s hair or skin, door knobs, laboratory bench, dust, reagents, ther- mocycler and pipette tips are common c sources of contamination. in stabilizing Ideally, a laminar air flow bench with filtered air provides a clean dust-free environment. 5 mol/L RFLP in and guani- RNA Sample preparation should be carried dinium ! isothio- out in a separate room or area. cyanate Recently, Neumaier et al. have published recommendations on quality assessment of molecular biology diagnostics (160) desribing preanalytical aspects:

64 Special aspects in molecular biology

Specimens: PCR analysis can be ap- blood for the extraction of DNA from plied to EDTA and citrate blood, dried leukocytes requires no special stabiliza- blood (filter-paper cards), bone, mar- tion. row, buffy coat, sputum, mouthwash, bronchial lavage, cerebrospinal fluid, Sample dispatch: samples stabilized ap- urine, stool, biopsy material, cell cul- propriately may be dispatched by post tures, fixed or embedded tissue etc.). at room temperature. This applies also Depending on the test material, a pre- to EDTA whole blood for DNA prepara- treatment of the sample may be neces- tion and GITC stabilized samples for sary prior to stabilization, e.g., lique- RNA recovery. Cooling is not neces- faction of sputum. sary, but depending on the application the prolonged storage at room temper- Sampling: sampling is best done in ature will result in a critical loss in sen- closed disposable sampling systems as sitivity. Samples are to be dispatched in are customary with other clinical test breakproof containers. Non-stabilized material. New disposable plasticware samples must be shockfrozen and than is considered to be free of nucleases. be dispatched in dry ice. The cooling When using nonclosed sampling sys- chain must not be interrupted. tems, at least disposable gloves are to be worn. Sample storage: specimen for DNA analysis are to be stored in 10 mM TRIS, Sample stabilization: stabilization of test 1 mM EDTA, pH 7.5–8.0 at 4°C. Speci- material is essential as nucleic acids mens for RNA analysis should be kept degrade rapidly. This is especially im- in buffered solution preferably at portant where RNA is to be analyzed. –20°C. GITC stabilized RNA samples The fast inactivation of DNases and may be stored for approx. seven days RNases is achieved reliably by chaotro- at room temperature. pic substances (here especially guanidinium-isothiocyanate, GITC). Organic In conclusion, understanding the prean- solvents, e.g., phenol, may be added in alytical pitfalls in molecular biology ap- parallel. Extraction systems with those plications and minimizing or elimina- additives are commercially available, ting them is a prerequisite to the Fig. 27-3 e.g., RNazol, Trizol. The limited stability successful utilization of such techniques β Microchip PCR as of reducing substances (here: -mercap- (95, 119, 143, 160, 190). diagnostic tool toethanol) and their effect on sample stability is to be considered. The user has A A A A C C C to be aware that batches of ready-to- A A A A G G G A A A A G G G use extraction solutions have limited A A A A T T T shelf life due to instability of single com- C C C C C C C ponents, e.g., β-mercaptoethanol. En- G G G G G G G Nucleotides A A A A C C C riched cells or native specimens of sin- C C C C C C C gle components are lyzed by addition A A A A G G G C C C C C C C of GITC. The final concentration of GITC 10 10 10 10 10 10 10 in the stabilized sample should not fall n n n n n n n lower than 4 mol/L. Material stabilized } Spacer in this manner needs not to be cooled. Carrier At temperatures lower than room tem- A = Adenine C = Cytosine G = Guanine T = Thymidine perature, GITC may crystallize. EDTA

65 When gases evaporate

Preanalytical variables in blood gas, and pH is decreased due to respiratory acid-base and electrolyte measurement and metabolic components along the have recently been addressed in IFCC arteriovenous blood flow. Arterial and recommendations (30). capillary sampling sites should therefore be preferred to obtain systemic acid- base and blood gas results. If in- Anticoagulant dwelling catheters or cannulae are Heparin is the recommended anti- used for sampling, it has to be ensured coagulant for blood gas and electrolyte that fluid or flush solutions are removed determinations in whole blood. Dry completely from the system by with- sodium heparinate may increase sodium, drawing a volume equal to three times decrease pH, bicarbonate and base the volume of the catheter system prior excess. In addition, ionized calcium is to blood collection (151). Blood should decreased if binding sites on heparin be collected anaerobically to prevent are not saturated. The recommended gas exchange with the surrounding air. final heparin concentration in blood dif- Venous occlusion by tourniquet should fers for glass and plastic tubes (195). be restricted to a maximum of 2 minutes. Formation of bubbles should be prevented. The sites recommended for sampling blood are those described in the chapters on arterial and capillary Recommendations on the use sampling (see p. 20–23). of heparin in blood gas and electrolyte analysis Recommendation on capillary Heparin solution may be added at sampling a final concentration of 8–12 IU/mL When sampling from skin, the first in glass and 4–6 IU/mL in plastic drop should be removed and blood tubes. The final concentration allowed to flow into the he- ranges for buffered heparin solu- parinized capillary without any tions should be, in mmol/L: squeezing. The tip of the capillary Sodium: 120–150 should be placed deeply into the Potassium: 3.5–4.5 drop and filled completely without Ionized calcium: 1.2–1.4 any pressure while holding in a Chloride: 100–130. horizontal or slightly downward position. The capillary in the drop The pH of the heparin solution should be closed off immediately should be between 6 and 8 (30). with a plastic cap followed by in- sertion of a metal mixing bar (”flea“). The opposite end of the Specimen collection There is a difference in blood gases capillary should then be sealed. and acid-base status between arterial Blood and heparin are mixed by and venous blood due to metabolism in moving the bar with the aid of a the respective limb or tissue. In general, magnet from end to end five times.

oxygen is consumed, CO2 is increased

66 Special aspects for blood gases and ionized calcium

∆ Storage and transport pO2 (kPa) (mm Hg) Specimens for blood gas and elec- 4.8 36 trolyte measurements may be affected during storage by the following pro- 30 plastic syringe cesses (133): 3.2 24

18 ● Metabolism of blood cells: glycolysis, mainly in red cells, causes formation of 1.6 12 lactic acid and shifts in pH, bicarbo- 6 0.4 glass syringe nate and base excess towards the 0 range of metabolic acidosis. Oxygen -6 consumption in leukocytes and platelets –0.8 decreases pO2 and increases pCO2. 0 10 20 30 40 50 t (min)

The fall in pO2 is accelerated if the original sample pO is elevated. The 2 Fig. 28-1 metabolic processes can be reduced gested that storage in ice water should Alteration of pO in by cooling the sample. Cooling is not exceed 30 min when stored in 2 whole blood (pO2 = necessary if the sample cannot be ana- plastic. Glass capillaries and glass 85 mm Hg ^= 11.3 kPa) lyzed within 15 min of sampling. syringes remain gas-tight for several stored in a plastic or hours (133) (Fig. 28-1). glass syringe for 45 ● Ion release from blood cells: pro- min at room temperature (mean and SD of longed storage, vibration during sample Sample preparation transport and severe thrombocytosis 15 measurements in are factors which may contribute to an Upon arrival of samples for blood gas each type of syringe) increase in potassium and a decrease and acid-base measurement, careful (133) in ionized calcium in the plasma. remixing of the samples prior to analysis During the first hour of storage in ice is necessary. water, the average increase in plasma potassium concentration is 0.1 mmol/L Specimens in glass capillaries should (133). be remixed by moving a metal wire from end to end for 5 to 10 sec. Glass Ionised calcium can be stabilised up to or plastic syringes should be inverted 8 hours after separating plasma by 10 times and then rolled horizontally centrifugation (96). for 10 sec (30). No bubbles or dead space should be formed during mixing. ● Gas exchange: As mentioned above, Mixing the sample is of special impor- plastic materials are not absolutely gas- tance if haemoglobin is measured si- tight. It has been observed that refrig- multaneously (134). erated plastic tubes leak more gases than those kept at room temperature. A compromise has therefore been sug-

67 The right time for drugs…

Fig. 29-1 concentration in blood Sampling – a matter of timing The elimination of 8 The optimal sampling time varies with most pharmaceuticals takes place in accor- the drug and the dose schedule used 2 dance with a first-order (see Tab. 29- and Fig. 29-2). reaction, i.e. a certain percentage of the total 4 When making an estimate of a quantity in the body is meaningful interval to select between eliminated per unit of 2 two determinations, it should be time irrespective of the remembered that approximately five 1 administered dose and 0.5 half-lives are necessary for equilibrium the concentration of the to be reached in the body between pharmaceutical agent 0481216202428 time of day intake and elimination, i.e. before in the sample (167) adapting the dosage one should wait until at least this interval has elapsed Which sample for TDM? before the next measurement of the (49, 124, 136) drug concentration. Maxima are given Fig. 29-2 in Tab. 29-2 , minimum concentrations Concentration of theo- The most commonly sampled body flu- should be obtained shortly before the phylline in plasma af- ids are blood, plasma and serum next dose is applied (Tab. 29-2 ). ter oral administration because a good correlation between of pills either as imme- drug concentration and the therapeutic Tab. 29-1 Rules for blood sampling in TDM diate-release formula- effect can usually be found. In addition, tion (red points) or as Time at which blood sample should be taken TDM in plasma/serum is useful in sustained-release for- Long-term therapy Basically always in the steady-state mulation (blue points). preventing toxic side effects of the drug (after approx. 5 half-lives) The immediate-release by overdosage or decreased elimina- formulation was ad- tion (metabolism). Intravenous One must wait until the distribution ministered 3 times a administration phase is completed (approx. 1–2 day (at 6.00, 15.00 In special cases, urine is also useful as hours after completion of the and 22.00 h), the sus- a sample material; saliva and CSF are infusion) tained-release formula- analyzed less frequently, although they Exception: tion 2 times a day at may better represent the free drug Digoxin 6 – 8 hours 8.00 and at 20.00 h concentration in some cases. (53) Digitoxin 6 – 8 hours

concentration in plasma (mg/L) For further details, see the NCCLS- 15 document on therapeutic drug monitoring (148).

10 Which specific aspects of sampling are important for TDM? Blood should not be taken from the arm 5 into which drugs or transfusion fluids are being infused.

0 Heparin, EDTA, or potassium oxalate 00 00 00 00 00 8 12 16 20 24 can be used as anticoagulants. How- time of day ever, heparin leads to a change in

68 Special aspects in therapeutic drug monitoring (TDM) protein binding in some drugs (236). To ensure their integrity, the samples EDTA is believed to be the optimal anti- should be sent to the laboratory without coagulant and should be the agent of delay. To avoid haemolysis and decom- choice for measuring levels of tricyclic position, only plasma, serum, urine or antidepressants since, by chelating CSF should be sent. Nitrazepam, chlor- divalent cations, EDTA might contribute azepam and cocaine, for example, to the stability of these drugs by protect- have been found to decompose during ing them from oxidation (147). storage of blood samples at 4°C.

Haemolyzed blood samples are pre- For ciclosporine A (CsA) which parti- ferred to serum or plasma for cy- tions into the red blood cell rapidly closporine determination, because nu- when blood cools from body tempera- merous factors (temperature, haema- ture (37°C) to room temperature (20°C) tocrit, concentration of lipoproteins) af- after collection, whole blood is the fect the blood/plasma relationship specimen of choice. The preferred anti- of ciclosporine (238). EDTA is also re- coagulant for monitoring CsA is EDTA commended as an anticoagulant. (192).

Some immunoassays for drugs can be When plasma is separated at room disturbed by unspecific cross-reactions temperature, the plasma to blood ratio of endogenous interference factors, for CsG is 0.8, whereas for CsA it is e.g. digoxin-like immunoreactive sub- 0.6 (147). stances (steroids, lipids) may interfere with digoxin (falsely elevated results). How stable are samples at Sampling of urine should be carried different temperatures? out over at least 7–10 biological half- Stability of samples for TDM is summa- lives (see Annex and (52)), which covers rized in the “List of Analytes“ (see more than 99% of the excretion phase. annex). Strict observation of the kit producer‘s instructions is recommended. Sampling times for saliva should be similar to those for blood. The mouth The freezing of blood for the deter- should be thoroughly cleansed with mination of ciclosporins should be water before sampling. The saliva must strictly avoided. In addition, samples be completely free of food particles for fluorouracil need to be collected on and any drug retained in the mouth ice (unstable at room temperature). following oral administration. Frozen samples should preferably be thawed at room temperature. Too rapid thawing by warming the sample may What about storage and transport? cause overheating and decomposition. Disposable containers should be used Thorough mixing is very important in to collect the specimens in order to order to secure homogeneity prior to reduce the possibility of contamination. analysis. Some plasticizers released from plastic syringes or from the rubber closures of glass containers may affect assay results (191).

69 The right time for drugs…

Tab. 29-2 Therapeutic Drug Monitoring: Pharmacokinetic properties with recommendations for sample collection. Data for adults (52, 74, 187). Substance Time to reach Time to reach Elimination Protein binding Recommendation on sample maximum steady-state half-life collection concentration ANTI-ARRHYTHMIC AGENTS Amiodaron 3–7 h (oral) 4 h – 25 d > 90% 15 min (i.v.) (single dose i.v.) 7 h – 80 h (oral) at the time of steady state: 20 – 100 d Quinidine 1–3 h 2 d 6–7 h 80–90 % Maximum about 8 h after administration of sustained-release preparations Disopyramide 2–3 h 1–2 d 4–9 h 10–65 % Protein binding is concentration- dependent Lidocaine End of the 30–90 min 70–200 min 60–70 % During the infusion; protein binding is initial dose concentration-dependent. Formation of active metabolite Procainamide/ 1–4 h (oral) 15–25 h 3–5 h approx. 15 % Maximum immediately after the last N-acetyl-procainamide (NAPA) 15–30 min (i.v.) 6–10 h orally administered dose; oral absorption is very variable ANTIBIOTICS Gentamicin* 1 h after i.m. < 30 y of age 1.5–6 h Sampling time 1 h after administration Tobramycin* administration; 2.5–15 h 0.5–3.0 h Trough level just before subsequent ≤ 10 % Netilmicin 30 min after i.v. > 30 y of age 2–3 h injection Amikacin administration 7.5–75 h 1.5–15 h Vancomycin 30 min 20–30 h 4–10 h 30 –55 % Peak 30 min after infusion of 1 h Streptomycin 1–2 h 10–15 h 2–3 h ≤ 30 % Peak 1– 2 h after i.m. infusion ANTICONVULSANTS Carbamazepine 6–18 h 2–6 d 10–25 h 65–80 % During the dosage interval; actual Clonazepan 1–2 h 20–60 h 83–87 % sampling time unimportant Ethosuximide 2–4 h 7–14 d 10–60 h 0 % Phenobarbital 6–18 h 10–25 d 50–120 h 50 % Actual sampling time unimportant Phenytoin 15–30 h 8–50 d 25–200 h 92 % During the dosage interval; actual Sustained-release preparations 3–9 h sampling time unimportant Primidone 0.5 –7 h 2 – 4 d 6–8 h 35 % Phenobarbital 10–25 d 48–120 h ▲ ▲ Valproic acid 2–8 h 2–3 d 8–15 h 90 %

70 Special aspects in therapeutic drug monitoring (TDM)

Substance Time to reach Time to reach Elimination Protein binding Recommendation on sample maximum steady-state half-life collection concentration

BRONCHOSPASMOLYTIC AGENTS Theophylline 1–4 h 2 d 3–12 h 55–65 % Maximum approx. 4 h for sustained- release preparations CARDIAC GLYCOSIDES Digitoxin 3–6 h 30 d 6–8 d 90–97 % 8 to 24 h after ingestion Digoxin 60–90 min 5–7 d 40 h 20–40 % 8 to 24 h after ingestion IMMUNOSUPPRESSANTS Ciclosporine A 2–6 h approx. 2 d 10–27 h 90 % Immediately before the next dose Tacrolimus 1–2 h 3 d 6–21 h 99 % Use EDTA whole blood (liver transplantation) 4–57 h (kidney transplantation) PSYCHOPHARMACEUTIC AGENTS Amitriptiline 2–6 h 3–8 d 17–40 h 90 % Desipramine 2–6 h 2–11 d 12–54 h 75–90 % Non-critical. In equilibrium, just before Imipramine 1–6 h 2–5 d 9–24 h 63 –95 % next dose is taken Nortriptiline 2–6 h 4–20 d 18–56 h 87– 93 % Lithium 1–3 h 3–7 d 14–33 h 0 % 12 h after the last administration CYTOSTATIC AGENTS Methotrexate 1–2 h 12–24 h 2–4 h 50–60 % Varies from person to person

* In newborns the biological elimination half-life is about 8 h (187)

71 Bacteria and viruses

The diagnostic value of a microbiologi- biological testing than swab specimens. cal test is influenced by a number of Aspirates are delivered to the laborato- pre-analytical factors (129, 184). At- ry in the syringe after the needle has tention must be given to the following: been removed and the syringe securely capped. In the case of open wounds, ● Unambiguous diagnostic question the superficial secretions should be ● Precise details of the collection site wiped off to remove interfering secon- ● Correct method and time of collec- dary organisms and a swab specimen tion then collected from the margins of the ● Suitable transport systems wound. Swab specimens must be pro- ● Shortest possible transport times tected from drying out during transport. ● Correct storage of the specimens This can be done by placing the swab before processing either in a liquid broth or in a transport medium. In the case of low bacterial Various containers for the collection counts the volume of the specimen should and transport of specimens for microbi- be as large as possible. Specimens for ological testing and the requirements blood cultures if possible should be col- for such containers are summarised in lected while the fever is rising. If infec- Fig. 30-1 and Tab. 30- 1 . tive endocarditis is suspected up to ten blood cultures must be collected. Even the most sophisticated transport system can never be a substitute for Short transport times and variable trans- short transport times and immediate port or storage temperatures are impor- specimen processing. tant for several reasons. Refrigeration and pH changes of the specimen or ex- Tab. 30-1 : Requirements for specimen posure to oxygen reduce the survival containers for the transportation of times of a number of organisms such as microbiological specimens meningococci, gonococci, Haemophilus, 1. Sterile pneumococci, Bordetella, Salmonella, 2. Non-corrosive Shigella, cholera vibrios, Helicobacter 3. Sufficiently large pylori and anaerobic organisms. While 4. Secure closure the viability of these environmentally 5. Unbreakable sensitive organism can decrease rapid- 6. Transport media ly during the transport, others multiply if the transport times are too long. This without growth enhancers or makes the quantitative evaluation of a with culture media for certain organisms culture difficult (urine). However, the sought organism can also become over- Bacteria grown with other organisms. All speci- When collecting specimens for bacterio- mens for bacteriological testing should logical testing particular care is to be therefore be delivered to the laboratory taken to avoid contamination. Before within two hours after collection. The trans- aspiration the skin must always be port and storage requirements for bac- meticulously disinfected. Purulent le- teriological testing are summarised in sions should be aspirated through the Tab. 30- 2 . If these conditions cannot skin, if possible, as this is easier to dis- be met, inoculation of a culture bottle is infect than the mucous membranes. Liq- recommended or, for urine samples for uid material is more suitable for micro- example, the use of dipslides.

72 Special aspects in microbiology

Tab. 30-2 : Transport and storage conditions for various specimens for bacteriological testing (29, 129) Specimen Transport Storage temperature Blood Blood culture bottle Room temperature or 37 °C Abscess material Short transport times: leave Room temperature, do not Cerebrospinal fluid specimen in (capped) syringe incubate, protect from cooling Pleural, pericardial, under anaerobic conditions. peritoneal, synovial fluid Delayed transport: use Paranasal sinus secretions transport medium Bronchoalveolar lavage (BAL) fluid Prompt transport (2–3 h) Cool Sputum, other secretions Stool Urine Dipslide Room temperature or 37 °C Swab specimens from Eyes Ears Mouth Throat Swab in transport medium Room temperature, Nose (> 4 h transport time) do not incubate Urethra Cervix Rectum Wounds Biopsy material Prompt transport in sterile Cool isotonic saline

Fungi (130) to the laboratory promptly in a sterile container. The same applies to the de- To obtain skin specimens for mycologi- tection of yeasts or moulds in sputum cal testing, scrapings from the active specimens with morning sputum speci- areas of the lesions, i.e. the edges, are mens being preferred. Tissue speci- collected with a scalpel after thorough- mens for mycological, as for bacterio- ly disinfecting the skin area. Skin, hair logical, testing should be placed in (collected from the edges with epilation isotonic saline and sent to the laborato- pipettes or excision off in the case of ry as rapidly as possible. For mycologi- deposits on the hair) and nail clippings cal testing of the vagina, the upper res- should be sent to the laboratory dry in piratory tract or the stool, submission of sterile containers. Scrapings from the two swab specimens in sterile contain- underside of the nail are used for cul- ers is recommended. Specimen trans- ture. For detection of yeasts in urine, a port at room temperature is usually un- random urine specimen should be sent critical for mycological cultures if the

73 Bacteria and viruses

Tab. 30-3 : Specimen collection, processing and transport for parasitological tests (I) = Immediate examination (92) Specimen material Specimen type and transport Parasites (direct and indirect detection) Parasite itself or Isoton. NaCl (endoparasites) e.g. Ascaris, proglottides components of parasites 70% alcohol (ectoparasites) e.g. fleas, lice Stool for transport Stool tube Eggs or larvae of intestinal For Lawless-stain fix in nematodes, cestodes, intestinal

alcohol sublimate (alc./HgCl2) trematodes, liver trematodes, lung trematodes. Cysts of protozoa: amoebae, flagellates, ciliates, coccidia, microsporidia. Vegetative forms of protozoa (particularly amoebae, Giardia) Stool for immediate At room temperature for direct Vegetative forms of protozoa examination examination (I) (particularly amoebae, Giardia) Duodenal fluid At room temp. for direct examination (I) Vegetative forms of Giardia Urine 24-hour urine Schistosoma haematobium Blood Thin film, thick film, heparinised blood Plasmodia, trypanosomes, microfilariae Bone marrow Smear, sterile bone marrow Leishmania Sputum Sputum tube Paragonimus eggs, larvae of intestinal nematodes, in some cases Echinococcus hooklets Skin Skin snip in isoton. NaCl (I) Onchocerca (microfilariae) Sterile skin biopsy Leishmania Recovery of eggs or Cellulose tape technique Pinworms adults from perianal skin

transport times are short. In the case of iates, helminths, cestodes), tissue speci- long transport distances refrigeration of mens of the affected organs (Trichinella the specimens (not necessary for swab spiralis larvae, Echinococcus) or the specimens) is recommended in order to parasites themselves (arthropods: ticks, prevent overgrowth of the slowly grow- mites, insects). In the case of stool spec- ing fungi with bacteria. If a phycomy- imens it should be noted that the vege- cete infection is suspected (e.g. Mucor) tative stages are unstable. These stages rapid specimen transport without refrig- can only be detected by examination eration is necessary. of fresh, body-warm stool. Cysts are stable. MIF (merthiolate-iodine-formalin) and SAF solution (sodium acetate – for- Parasites (92) malin) have become routine for para- Specimens examined for the diagnosis site concentration and preservation in of parasitic infections are blood (plas- stool specimens. For most specimens modia, trypanosomes, leishmaniae, mi- transport is uncritical and special trans- crofilariae, Loa loa), stool (Giardia, cil- port conditions need not be observed.

74 Special aspects in microbiology

Refrigeration of the specimens is rarely Tab. 30-4 : Cases in which the specimen should ever necessary. Arthropods are sent to only be processed after consultation the laboratory in 70% alcohol. Tab. with the requester (129) 30- 3 gives a brief overview of preana- 1. No label/identification lytical factors important for parasitolog- 2. Abnormally long transport time ical testing. 3. Unsuitable or leaking containers 4. Unsuitable specimen A fundamental prerequisite for a para- 5. Submission of the same specimen material with the sitological examination is a travel histo- Fig. 30-1 same question or request within 24 hours Containers for trans- ry (place, time and duration of visit) (except blood samples) port of specimens for and information on onset of symptoms, microbiological studies treatment and whether or not the patient is immunosuppressed. A request stating “test for parasites” is inadequate.

Viruses The time of specimen collection for virus isolation and identification is critical. Usually the material should be collected immediately after the onset of symptoms (if possible within the first three days). Swab tube Swab set with trans- Blood culture As a general rule, specimens should be port culture medium bottle delivered to the laboratory rapidly at 4°C in an insulated container. Viruses usually remain stable for 2 to 3 days under these conditions (130). Swab specimens (nose, throat, eyes), pharyn- geal washings, vesicular fluid from skin lesions, stool, urine and CSF are used for analysis.

Specimens for microbiological studies which do not meet certain standards Sputum Specimen tube for detection of CSF/Urine/Aspirates (see Tab. 30- 4 ) should either be reject- tubercle bacilli ed or only analysed after consultation with the requester.

Tracheal aspiration set Stool

75 Can turbid samples be used?

The lipemic sample crons. In contrast, a more homogene- Plasma and serum samples are some- ous turbidity is in most cases caused by times turbid to varying degrees due to the presence of increased concentra- an increased lipoprotein content (Fig. tions of VLDL. 31-1). In nearly all cases, turbidity is caused by an increased triglyceride The concentration of triglycerides lea- concentration. The turbidity may be ding to turbidity depends on the com- slight, often called opaque, translucent, position of lipoproteins. Chylomicrons, turbid or milky. The degree of turbidity due to their size, deflect light at de- depends not only on the amount of tectable rates even at triglyceride con- Fig. 31-1 triglycerides but also more markedly on centrations below 300mg/dL (3.4 Plasma samples with the presence of macromolecular spe- mmol/L). However, the intermediate different degrees of cies of lipoproteins. Turbid samples are and low density lipoproteins can be in- turbidity therefore called lipemic. visible even at triglyceride concentration of 800mg/dL, or higher. Varying degrees of turbidity are observed with VLDL, depending on their size and composition (10, 60).

Relevance of turbidity as an interference factor Whereas the degree of hyperlipidemia is of diagnostic relevance, the interference of lipoproteins with the determination of lipids and other blood constituents should be regarded as disturbing interference factors which should be avoided as far as possible. The diagnostic importance of turbidity Because normal samples do not exhibit Mechanisms of interference any turbidity except after a fatty meal, The following mechanisms have been the turbidity of a sample is always of found to cause either falsely low or clinical relevance and should be as- high laboratory results: sessed, documented and reported by ● Inhomogeneity: Triglyceride-rich lipo- the laboratory (73 and Annex). It may proteins cause them to float during cen- indicate hypertriglyceridemia due to an trifugation and storage of serum/plasma increase in chylomicrons, very low den- samples. When analyzed after such sity lipoproteins (VLDL) or both. As de- treatment (centrifugation) without careful scribed in textbooks, these forms can mixing, triglycerides and other constitu- be differentiated by observing the float- ents may be inhomogeneously distri- ing of lipoproteins during centrifuga- buted in the sample. This may cause a tion and storage (31, 66, 213). A dis- disproportionately high concentration tinct creamy layer floating over a clean of lipids in the upper layer and cause layer after centrifugation on storage interference in other methods like total over at least 12 hours in the refrige- protein. On the other hand, lipids may rator indicates the presence of chylomi- displace water in the upper phase of a

76 Samples: From the Patient to the Laboratory. W. G. Guder, S. Narayanan, H. Wisser, and B. Zawata Copyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-30981-8 Effects of lipemia

Fig. 31-2 sample thereby leading to a lower ap- relative Method – dependent parent concentration of water-soluble concentration inferference of various components like electrolytes and meta- 2.0 uric acid analyte determinatins bolites. by increased 1.5 protein, glucose triglycerides (65) ● chloride Water displacement is also respon- 1.0 sodium creatine kinase sible for the higher concentration of creatinine sodium and potassium observed in 0.5 direct ion-sensitive electrode measurement compared to flame photometry 0 60 120 240 480 960 (110). In exceptional cases, lipids can triglycerides added mg/dL displace up to 10 % of the water content of a serum/plasma sample. and the analysis repeated with the ● Interference by turbidity: Photometric clarified sample. procedures are sensitive to turbidity at nearly all wavelengths (65). This leads Care has to be taken regarding the to various degrees of absorption (Fig. clarification procedure itself as a pos- 31-2). sible cause of interference.

● Interference by physicochemical me- In some cases a change in methodolo- chanisms: Lipoproteins in the sample gy may be helpful in eliminating inter- may incorporate lipophilic constituents, ference due to lipids. Thus, a second thereby decreasing their accessibility to wavelength may compensate for turbidity. antibodies. Likewise, electrophoretic and Alternatively, a blank sample may be chromatographic procedures may be run without the relevant reactant but disturbed by lipoproteins. under otherwise identical conditions. In each case the degree and type of turbidity should be documented and reported “Diagnosing” and “treatment” of and an aliquot of the untreated sample interference due to turbidity stored for later testing for verification purposes. Relevant turbidity can easily be detected with the naked eye. Alternatively, Procedures for treating lipemic samples the turbidity of each sample can be should be documented in the quality as- measured by automatic analyzers using surance manuals of each laboratory. a specific wavelength (660–700 nm) Producers of test kits should indicate (65). The degree of interference of that interference by lipemic samples each method can be quantified by has been tested for and provide respec- adding different amounts of sample tive information in the product leaflet from a hyperlipemic patient to a clear (73 and Annex). sample subsequent to analyzing the concentration of both samples separately. When a test is known to be disturbed by any of the mechanisms mentioned, triglycerides may either be removed from the sample by ultracentrifugation (14) or precipitation (114)

77 A difficult case

Fig. 32-1 Cold agglutinins ues are low, resulting in high MCH and “MCV”-determination Antibodies as interference factors in MCHC values. The leukocyte and of blood in cold clinical chemistry are often neglected thrombocyte counts are falsely eleva- agglutinin disease at different temperatures since detection of this factor is difficult ted, because the agglutinates depen- (15) under day to day routine conditions. ding on their size are either counted in Analytical procedures in clinical chem- the leukocyte or thrombocyte channel. The blood smear shows agglutination 100 of erythrocytes. Both the determination of blood group and crossmatching pro- 75 cedures can be affected by cold agglutination in two ways. First, panaggluti- nation introduced by antibodies can 50 ° 20 C influence the correct assignment of 31°C 36°C blood group antigens and the cross-

frequency (%) 25 37°C matching procedure. Second, the cold agglutination antibodies may mask other types of antibodies which may 30 60 90 120 150 180 210 affect analytical procedures in a differ- cell volume (fL) ent manner.

istry, haematology and immunohaema- Cryoglobulins tology can be affected by antibodies (107). Antibodies may affect the cell Cryoglobulins crystallize in samples count of erythrocytes, leukocytes and kept at room temperature. The resulting Fig. 32-2 platelets (234). High titers of cold ag- particles are of varying shape and may Distribution of cryo- glutinins directed against erythrocytes mimic leukocytes, resulting in a falsely globulin particles at lead to agglutination. Such agglutina- elevated leukocyte count (Fig. 32-2). different temperatures tion alters the electronic cell count in Moreover, high concentrations of cryo- and the corresponding increase in leukocyte the following way: erythrocyte count is globulins can affect erythrocyte count, count at different low at normal haemoglobin concen- haemoglobin determination (floccula- storage times at room trations; MCV is grossly enhanced tion phenomenon) and platelet count temperature (1) (Fig. 32-1); calculated haematocrit val- (pseudothrombocytosis) (Fig. 32-2). The blood smear shows flocculated dark particles G/L blue crystals without elevated leukocyte 8 numbers. The phenomenon of pseudo- 7 6 4°C 25°C 37°C leukocytosis depends on the length of 5 4 contact, temperature, concentration of 3 cryoglobulins and the interaction of 2 1 cryoglobulins with other plasma pro-

teins (1). This is valid for all cell number

3,7 3,7 5,8 9,2 9,2

5,8

2,9 7,3

4,6 determinations by particle counting.

14,6

18,5 11,6 particle diameter (µm) leukocytes G/L 50 EDTA-dependent antibodies 40 30 Antibody-induced falsely decreased 20 platelet counts (without haemorrhagic 10 123456 time (h) diathesis) can be due to cold agglutinins or antibodies active in presence

78 Pitfalls with endogenous antibodies of EDTA. In both cases, agglutination resis. Both types of macro CK may affe- takes some time. Thus, a prolonged ct accurate quantification of CK-MB by delay between obtaining the sample means of CK-M-inhibiting antibodies re- and platelet counting results in a more sulting in falsely elevated CK-MB activi- pronounced pseudothrombocytopenia. ties. Another example is macroamylase Platelets of patients with thrombasthe- which is characterized by enhanced nia, which lack the membrane glyco- activity in serum while urinary amylase proteins IIb and IIIA, do not react with excretion is unchanged (209). EDTA-dependent antibodies. This observation suggests that these glycoproteins are actively involved in binding the Autoantibodies antibody. Depending on their shape Immunoassays can be affected by au- and volume, thrombocyte aggregates toantibodies or heterophilic antibodies may be counted as leukocytes. In addi- (21). Well-described examples are au- tion to pseudothrombocytopenia elevat- toantibodies directed against triiodo- ed cell counts for leukocytes may be thyronine and thyroxine. Thyroid hor- observed. Detection of particles of the mone concentrations are apparently size of lymphocytes in the white cell enhanced since the tracer is bound not histogram is evidence of a spurious only to the receptor antibody added to count of leukocytes. Staining of peri- the sample but also to the autoanti- pheral blood allows detection of aggre- body. Antiphospholipid antibodies in gates of platelets. Other causes of plasma results in increased APTT values falsely decreased thrombocyte counts because the antibody binds phospho- are platelets adhering to leukocytes lipids used as reagent in the assay. (platelet satellism), giant platelets, or analysis of partly coagulated blood samples caused by a wrong sampling Heterophilic antibodies technique. Heterophilic antibodies are detected in some human serum samples, the mechanism underlying generation of these “Macroenzymes” antibodies being unknown. The possibility of complexes with immuno- In some cases, interference by hetero- globulins (macroenzymes) has been philic antibodies can be of diagnostic demonstrated for all diagnostically rele- significance. If antibodies have anti- vant enzymes. A consequence of such mouse specificity and assays employ- phenomena is an increased biological ing immunoantibodies from mice are half-life of such enzymes. The increased used (murine monoclonal antibodies), half-life may in turn result in enhanced interference of these assays is possible. enzyme activity which can provoke There are several reports in the literature further diagnostic measures. The pheno- describing wrong therapeutic measures menon of macroenzymes is primarily as a consequence of such antibody observed in elderly patients with chronic induced analytical errors (21). In this diseases. Well-described examples are case monoclonal antibodies caused the macro creatine kinase (CK) type I and interference. However, antibodies have type II. Macro CK type I is an immu- been described, which are unspecific noglobulin CK-BB complex. Type II re- and interfere as well. Only the latter presents polymers of mitochondrial CK, are classified as heterophilic antibodies which can be detected by electropho- in the narrow sense (116).

79 The serum sample looks reddish

Blood consists of cells and plasma. Haemolysis is not always accompanied Many constituents measured in plasma by the release of haemoglobin (for have relatively high concentrations in instance, if blood is stored at low, but blood cells (233). Therefore, haemoly- non-freezing temperature). Interferents sis should be avoided to obtain reliable may also arise from both platelet lysis results. and granulocyte lysis.

What is haemolysis? Mechanisms of interference The absence of red Haemolysis has been defined as the (73 and Annex) colour does not, how- ”release of blood cell constituents into The effects of haemolysis may be ever, exclude interfer- plasma/serum“. It is usually recognized classified according to the mechanisms ence by haemolysis, by a more or less reddish appearance involved: because haemoglobin is of the plasma/serum after centrifugation seen with the naked eye (Fig. 33-1), caused by haemoglobin ● Increase of intracellular constituents only at a level approx- imating 300 mg/L and released from the erythrocytes. As such, in the extra-cellular fluid. The efflux of higher. interference can occur even at lower intracellular constituents may occur in- concentrations of haemoglobin other- vivo, during sampling and at all stages wise invisible to the naked eye. of the preanalytical phase. According- ly, haemolysis may be a diagnostically

Fig. 33-1 relevant observation, defined as an in- Plasma samples with vitro influence factor when occurring various degrees of during sampling or other steps of the haemolysis preanalytical phase as it leads to alteration of the sample composition. Fig. 33-2 shows the effect of increasing Fig. 33-2 haemolysis on various serum analytes. Changes in various analytes with increas- ● Optical interference may be due to ing haemolysis in a the colour of the haemoglobin, which dual wavelength may change during sample storage routine analyzer due to haemoglobin formation. The direction and degree of interference 4.0 lactate dehydrogenase differs not only with the wavelength(s) 3.5 3.0 but also with the type of blank and 2.5 aspartate aminotransferase reagent used. Recently therapeutically 2.0 HDL-cholesterol applied artificial oxygen carriers based 1.5 alanine aminotransferase on haemoglobin structure (HbOC) have 1.4 1.3 potassium been introduced (32). This when ap- creatine kinase 1.2 plied in concentrations up to 50 g hae- 1.1 triglycerides moglobin/L create optical interferences 1.0 cholesterol urea, chloride, nearly indistinguishable from those cau- 0.9 magnesium, sodium

relative concentration (activity) 0.8 γ− sed by natural haemoglobin. 0.7 glutamyl transferase 0.6 0.5 ● Interference by intracellular consti- alkaline phosphatase, amylase 0 tuents with the reaction mechanism of 0 12345 the assay (chemical, biochemical and haemoglobin conc. g/L immunological interference). In this

80 Effects of haemolysis

case, a method-dependent interference total bilirubin fraction found in bilirubin assays studied is observed which is not due to optical 1.50 1.40 2,5-dichlorophenyldiazonium, interference by haemoglobin. Thus, detergent adenylate kinase released from blood 1.30 cells interferes with most standard 1.20 1.10 methods for the measurement of creatine direct reading method 1.00 kinase activity, the interference being 0.90 dependent on the concentration of the Jendrassik-Grof, Nosslin 0.80 inhibitors of adenylate kinase added to Jendrassik-Grof (+Fehling) 0.70 the reagent mixture (210). Fig. 33-3 0.60 illustrates the method-dependence of 2,5-dichlorophenyldiazonium, 0.50 detergent haemoglobin interference with various 0.40 2,4-dichloraniline diazo methods for bilirubin, caused by 2,5-dichlorophenyldiazonium 0.30 the peroxidative effect of the haeme nitrophenyldiazonium 0 (224). 1.0 2.5 4.0 5.0 haemoglobin g/L

How to ”diagnose“, prevent and stituents can be ascertained. Evaluation Fig. 33-3 ”treat“ interference brought about of the possible cause of haemolysis will Interference of haemo- by haemolysis (73 and Annex) certainly help to prevent interference. globin with various Overt haemolysis is easily detected In-vitro haemolysis can be prevented by diazo methods to measure bilirubin when the sample is visually controlled standardizing the preanalytical phase. (from (224)) before being analyzed. In-vivo and in- Use of standardized needles, closed vitro haemolysis may be differentiated tubes and calibrated centrifuges is of by comparing various samples of the great help in reducing haemolysis. The same patient and by analysis of sensitive use of plasma instead of serum can markers of in-vivo haemolysis such as also minimize haemolysis, especially haptoglobin and consideration of clinical by avoiding the release of cellular information. Consulting the clinician constituents from platelets (121). Fig. is advisable for any suspected in-vivo 13-2 (see p. 32) shows how differences haemolysis. In addition any unexpect- between serum and plasma potassium ed increase in ”sensitive“ analytes concentrations depend on the number should be regarded as arising from in- of platelets. vitro haemolysis unless this can be ex- The laboratory should be aware of the cluded. Free haemoglobin, lactate dehy- effects of haemolysis on specific tests drogenase (LDH) activity and potassium (201). The user should expect new should increase in parallel in this case. reagents and kits to be tested for the ef- Once diagnosed, results obtained from fect of haemolysis by the manufacturer a haemolytic sample should be with- and respective information given in the held (or sample not measured) if inter- product application manual. ference is to be expected. If a new Every laboratory should document how sample cannot be obtained, the clini- haemolyzed samples are to be handled cian should receive information about in their quality assurance manual. The the possible degree of interference responsibility of the laboratory for along with the result. A correction for- diagnostically reliable results can only mula as suggested by Caraway (33) be fulfilled by taking rigorous measures can be applied only if in-vitro haemoly- to prevent misinterpretation of results sis with parallel release of all con- caused by haemolysis (73 and Annex).

81 Does the laboratory have to know all my drugs?

Mechanisms of drug interference the bilirubin measurement by both the Interference with laboratory tests by above methods (208). drugs is so widespread due to the multi- plicity of drugs and laboratory pro- Another pharmacological effect relates cedures that a computerized directory to the ability of the drug to increase the is the best source of information avail- level of binding proteins. This can result able on the subject. In the main, how- in an increase in the level of analytes ever, drug interference on laboratory bound to such proteins. For instance, tests can be broadly categorized as be- oral contraceptives increase the plasma ing either biological or chemical. A concentration of thyroxine-binding pharmacological effect arises as a con- globulin, ceruloplasmin, transferrin and sequence of the drug being metabo- transcortin, thus increasing the level of lized in the body and the metabolite analytes bound (thyroxine, copper, iron subsequently interfering with the labo- and cortisol) (241). ratory test. Thus, while the parent drug propranolol does not interfere with the Drug interference that is classified as bilirubin methods of both Jendrassik- technical, on the other hand, relates to Grof and Evelyn Malloy, the metabolite in-vitro interference which could be 4-hydroxy propranolol interferes with either chemical or physical, such as

Tab. 34-1 Drug effects and interference; mechanisms and analyte changes Category Mechanism Example Analytes Change of drug in plasma

Biological Enzyme induction phenytoin γ-glutamyl transferase ➚ influence ➚ Enzyme inhibition in liver allopurinol uric acid in vivo

Enzyme inhibition in plasma cyclophosphamid cholinesterase ➚ Increased oral contraceptives copper ➚ binding protein (ceruloplasmin) Competing with novobiocin bilirubin, ➚ endogeneous com- unconjugated ponent for glucuronidation

Antivitamin warfarin, protein C ➚

effect phenprocoumon prothrombin ➚ Cytotoxicity liver biguanides lactate, ➚ kidney gentamicin alanine aminotransferase ➚ cis-platinum creatinine ➚ Chemical and Cross-reactivity spironolactone digoxin apparrent physical in immunoassays ➚ interference Chemical reaction cephalotin creatinine ➚ in vitro with Jaffe’ reagent ➚ Production of atypical salicylates haemoglobin A1 haemoglobins

82 Mechanisms and treatment of drug interference haemolyzed or icteric samples etc. 179, 218, 241). When using these (141). Tab. 34-1 lists some of the other lists, the method-dependence of many types of interference by the more com- of the effects described has to be kept in mon drugs on laboratory tests (98, mind. As with other types of interfer- 123, 141, 145, 241). ence, comparison of results obtained using two independent methods may indicate the mechanism of interference. Binding of drugs to protein Consulting with a clinician is advised in The binding of drugs to proteins can be order to prevent misinterpretation due to altered due either to the presence of drug interference. other drugs competing for the same binding site on protein or due to an increased level of fatty acids (141). In Sequence of sorting criteria general, drugs that are weakly bound tend to be displaced from their protein Dosage form Shape Colour binding sites by a competing drug or fatty acid. Thus, free drug levels can be white increased under such circumstances. sugar-coated round tablet Furthermore, unless the displaced drug yellow is rapidly metabolized, the patient orange would become toxic at the therapeutic level of dosage. red tablet oval pink Low albumin levels found in patients with liver and kidney disease affect violet protein binding. In such a case when blue multiple drugs are being administered, soft gelatin oblong competition for binding sites on albumin capsules green can be significant. For instance, when beige valproic acid is co-administered with phenytoin, the competition for protein brown binding sites can lead to the displace- hard gelatin triangular black ment of phenytoin by valproic acid capsules leading to decreased total phenytoin gray-silver coloured levels, since the displaced phenytoin is other forms rapidly transformed into an inactive metabolite (136).

The protein binding capacity of a par- Fig. 34-1 ticular drug can be dramatically al- Colour chart of drugs used to characterize tered in conditions such as in uremia and identify drugs where for instance the protein binding found of phenytoin can vary from 70% to near 0%.

The broad spectrum of drug interference with laboratory tests has been compiled in several reviews and books (123,

83 Everything under control?

Usually, the quality of a laboratory test individual needs in any given clinical result is assessed by defining the impre- situation. In contrast to the analytical cision and accuracy in comparison to result, which is often defined as a quality standards. These standards are “product“, the preanalytical phase may either obtained from experts, formulat- be defined as a mixture of processes ed through one‘s own experiences or and materials. Tab. 35-1 summarizes defined by external quality control numerous examples of materials and agencies (46, 221). No such standards procedures (processes) used during the exist for defining the quality of the pre- preanalytical phase. analytical phase. From present knowledge however, several criteria may be derived which may be taken as quality Who defines quality? criteria in each individual laboratory. Complaints, suggestions and proposals from persons involved are the major sources of information upon which a Defining quality quality assurance program for the pre- The aim of analyzing samples taken analytical phase can be initiated. The from patients is to obtain a result which quality of the products and processes describes the condition of the patient have to be defined in relation to the as represented by the analyte concen- medical needs. This can be done by a tration in blood or other body fluids. group of experts in a “quality circle“ or This result helps the doctor to arrive at by an external audit. Local, national or a diagnosis, providing it reaches him international standards and recommen- before he has to make a decision dations may also be used. The results of which may well be influenced by the these activities are published in the pre- result. Thus, the adequateness of the liminary quality manual, which, after request, the type of sample and the tim- evaluation, can be used as the basis for ing has to be defined in relation to the future quality assessment schemes. The

Tab. 35-1 Preanalytical processes and materials that can be subjected to quality assurance Step Process Materials Patient preparation Information on diet, posture and sampling procedures Urine containers Preparation of sampling Defining request, entering request, labeling tube Request form, request program, patient and sample identification system Sampling Identification of patient, timing, tourniquet, Needles, tubes, disinfectant cleaning site of sampling, selection of vein, artery or capillary sampling site, positioning of needle, changing tubes Transport Collecting samples, Sample containers, pneumatic transporting samples tube system, cooling systems. Sample treatment Registration, centrifugation, distribution, Identification and registration mixing, identification, extraction program Storage Timing of storage, selection of site Storage device, freezing and temperature, finishing storage, remixing device, temperature control after storage

84 Quality assurance in the preanalytical phase results of this evaluation and the objec- tives of the quality assurance program should be defined in a quality manual.

Quality of the sample

The adequateness of a sample should be addressed from the standpoint of patient, doctor and the laboratory.

Patient’s criteria: Painless sampling is preferable to painful sampling. Less blood is better than excessive blood collection. Recently a formula was recommended defining the optimal volume of sample considering technical and biological variables. If these rules are followed, the minimal amount of sample can be assured for the analysis ordered (see (71) and Annex). A rapid procedure of sampling is better than a drawn out procedure (i.e. spot or random urine sample versus 24 h urine except where it is absolutely necessary).

Doctor’s criteria: More information from one sample is preferable. Rapid sampling is preferred to time-consuming sampling. A rapid test is preferable to a Fig. 35-1a ▲ ▲ slow one. The ideal sampling procedu- Relative contribution re is one presenting a low degree of fore, it‘s timing is of critical importance of the preanalytical risk to both patient and phlebotomist. for the entire diagnostic procedure. phase to the total turn-around time of a diagnostic test Laboratory’s criteria: It is better to have Fig. 35-1a gives an example of the more sample than is needed rather prelaboratory and intralaboratory pre- Fig. 35-1b ▲ than an insufficient amount. An ad- analytical times analyzed (62, 70). Fig. Persons involved in equate amount has to be defined. A 35-1b shows that many different per- the preanalytical normal sample is preferred to a sample sons are involved; these have to be con- phase with potentially interfering (haemolytic, sidered when quality is to be improved. lipemic) or risky (infectious) factors. Standard sample size and anticoagulant to blood ratio is preferred to non- How can timing be documented standardized samples. and controlled? In order to clearly document the timing Quality of timing of laboratory testing, three essential times need to be monitored in assess- The preanalytical phase takes more ing quality: time than the analytical phase. There- 1. Time of sampling

85 Everything under control?

2. Time of arrival of sample in the cedures and aims of several preanalytical laboratory aspects of the laboratory, including con- 3. Time of printing of result sequences in cases of non compliance. This list also includes procedures on re- The difference between time 1 and 2 quests, sampling and handling details as gives the prelaboratory preanalytical well as regulations for transport to other time; the time interval between 2 and 3 laboratories (90). provides the intralaboratory preanalyti- A Working Group on Preanalytical cal plus analytical and postanalytical Quality has published recommendati- time. Further documentation of the vari- ons which can serve as a basis to de- ous phases of the preanalytical time is fine quality standards (71, 72, Annex). possible if analytical time is subtracted. By doing this, preanalytical phase was Fig. 35-2 Tab. 35-2 Contents of the quality manual for shown to be responsible for more than Documentation of pre- the preanalytical phase analytical times during 50% of the total turn-around time in 1. Request procedure a usual working day most laboratories. Fig. 35-2 gives an a. Forms b. Patient identification 2. Sampling Materials a. Needles b. Tubes c. Containers for non-blood samples Procedures a. Venous blood b. Capillary blood c. Arterial blood d. Timed urine e. Spot urine f. Cerebrospinal fluid example of the documentation of pre- g. Sputum analytical times in a laboratory infor- h. Ascites and pleural fluid mation system. i. Other samples 3. Transport The quality journal of the 4. Registration 5. Centrifugation preanalytical phase 6. Sample identification Procedures and standards used in the 7. Storage individual laboratory should be docu- 8. Handling of interference mented in a quality manual accessible a. Haemolysis to all employees as well as visitors and b. Lipemia 2 external quality managers. Tab. 35- c. Icterus gives an example of the possible table d. Drugs and containments of contents of such a quality manual. 9. Disposal of samples According to the ISO standard on quali- 10.Timing of the preanalytical phase ty management of medical laboratories 11.Documentation the quality manual has to contain a de- 12.Responsibilities tailed description on responsibilities, pro-

86 It is to be hoped that this book will help you to reach the gold standard in the preanalytical phase. References

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96 Reference Glossary

The definition of terms used in this analytical sensitivity: volume follows the “Vocabulary of The slope of the analytical calibration Reference Method Procedures and Ma- function. The term “analytical sensitivi- terials in Laboratory Medicine”, pre- ty” is not a synonym for detection limit. pared by R. Dybkaer (47). Other sources are given in brackets (26, 68, analytical specificity: 90, 100, 149, 156, 229). The ability of a measurement procedure to determine solely the measur- accession: able quantity it purports to measure. All steps necessary to ensure that a specific blood specimen and the bias of measurement: accompanying forms are unmistak-ably Bias is the difference between the identified as referring to a specific per- expectation of the results of measure- son. ment and the true value of the measur- and. accuracy: Closeness of the agreement between biological influence quantity: the result of a measurement and the See influence factor. true value of the measurand. biological reference value; additive: reference value: Substance, other than surface treatment Value of a measurement in an individ- designed to be irremovable, that is ual belonging to a defined reference placed inside the receptacle to facili- sample group of individuals. tate the preservation of the specimen, or is intended to react with the speci- The term “biological reference value” is men, in order to allow the intended not a synonym of the ambiguous term analysis to be performed (89). “normal value” as the individuals of the reference sample group may suffer analyte: from a defined state of disease. Component of a sample indicated in the name of a measurable quantity. component: Definable part of a system. analytical interference: In analytical chemistry the components Systematic error of measurement cau- of a system are sometimes divided into sed by an analytical interferent (90, “analyte”, “concomitants”, and 149). “solvent”; the latter two are often called “matrix”. analytical portion: The portion of material taken from the detection limit; limit of detection: analytical sample and on which the The minimum detectable value. Result measurement of the appropriate mea- of a measurement by given measure- surable quantity is actually carried out. ment procedure for which the probability of an analytically false negative analytical sample: result is b, given the probability a of an A sample prepared from the laboratory analytically false positive result (IUPAC sample and from which analytical por- recommends default values for a and b tions may be taken. equal to 0.05.)

diagnostic sensitivity: measurement. Inaccuracy of a meas- See nosographic sensitivity. urement describes a combination of systematic effects and random effects diagnostic specificity: that contribute individual components See nosographic specificity. of error of measurement.

endogeneous: influence, influence factor: Any factor or mechanism acting or de- Biological (in vivo and in vitro) influ- rived from the system from which the ence on the concentration of a mea- analytical sample is taken. See also in- surand in a system (i.e. venous blood). terference factor. interference: error of measurement; error: Systematic error of measurement cau- Result of a measurement minus a true sed by a sample component which value of the measurand. does not by itself produce a signal in the measuring system (CEN). The effect exogeneous: of a substance upon any step in the de- Any factor or mechanism added to the termination of the concentration or cat- sample either in vivo (i.e. drug) or to alytic activity of the analyte. the sample in vitro (i.e. contaminant). interference factor: good laboratory practice; GLP: Substance or component of the matrix Organization process and the condi- of a sample which differs from the ana- tions under which laboratory studies lyte and interfering with the analytical are planned, performed, monitored, procedure to give a false measuring recorded, and reported. signal.

imprecision of measurements; Interference factor is called influence imprecision: quantity by Dybkaer (47), defined as Dispersion of independent results of the measurable quantity that is not the measurements obtained under speci- measurand, but that effects the result of fied conditions. the measurement.

Imprecision of measurements, when ap- inter-individual variation: plied to sets of results of measurements, Distribution of the values within indi- depends solely on the dispersion of viduals of a given set. random error of measurement and does not relate to a true value of the internal quality control: measurable quantity. Imprecision is usu- Operational techniques and activities ally expressed numerically as the re- within a production site that are used to peatability standard deviation, an inter- fulfil requirements for quality. mediate precision standard deviation, or a reproducibility standard deviation international system of units; SI: of results of measurements. Coherent system of units of measurement adopted and recommended by inaccuracy of measurement; the General Conference on Weights inaccuracy: and Measures (CGPM). The SI is pre- Discrepancy between the result of sently based on seven base units of measurement and the true value of a measurements: metre (m), kilogram

98 Glossary

(kg), second (s), ampere (A), kelvin (K), Clinically true positive classifications di- mole (mol), and candela (cd) (229). vided by the sum of clinically true positive class plus clinically false negative intra-individual variation: classifications. Distribution of the values in a given individual. It is usually assumed that the nosographic specificity; variation occurs with time as an inde- not diagnostic specificity: pendent variable. Number of persons correctly classified by the results of measurement as not matrix: being in a defined state divided by the All components of the material system number of all persons not in the de- except the analyte. fined state. matrix effect: Clinically true negative classifications Influence of a sample property, other divided by the sum of clinically true than the measurand, on the measure- negative class plus clinically false posi- ment and thereby on the value of the tive classifications. measurand. patient sample (specimen): measurable quantity; quantity: An aliquot of a specimen that has been Attribute of a phenomenon, body, or appropriately collected, transported substance that may be distinguished and processed in the laboratory to pro- qualitatively and determined quantita- vide material for a specific laboratory tively. test (153 – 159). measurand: preanalytical phase: measurable quantity subject to mea- synonymous with pre-metrological surement. phase and pre-examination phase, starting from the request, including the measurement: examination requisition, preparation of Set of operations having the object of the patient, collection of the primary determining a value of a measurable sample, transportation to and within quantity. the laboratory, and ending when the analytical examination procedure measurement procedure: starts. Procedures included herein are Set of operations described specifical- called pre-examination procedures ly, used in the performance of measure- (89). ments according to a given method of measurement. precision of measurements; precision: metrology: Closeness of agreement between inde- Science of measurement. pendent results of measurements ob- nosographic sensitivity; tained under stipulated conditions (ISO not diagnostic sensitivity: 3534-1). Number of persons correctly classified quality assurance: by the results of measurement being in All the planned and systematic activi- a defined state divided by the number ties implemented within the quality sys- of all persons in that state. tem, and demonstrated as needed, to

99 Glossary

provide adequate confidence that an reference interval, reference values: entity will fulfil requirements for quality Do not use normal values because of (ISO 8402-3.5). In the clinical labora- the inherent ambiguity of the word tory sciences, it is customary to consid- “normal”. The reference interval defi- er internal quality control and external nes the 95% range of reference values quality assessment as complementary obtained from a reference population but not complete parts of quality assur- (199). ance. The term “external quality assurance” is also being used to comprise reference limit: the actions ensuring the transferability Upper or lower limit of reference of results of measurement. interval, not identical with clinical decision limit. quality system: Organisational structure, procedures, reference material: processes, and resources needed to im- Material or substance, one or more of plement quality management (ISO whose property values are sufficiently 8402-3.6). homogenous and well established to be used for the calibration of a mea- quantity: suring system, the assessment of a mea- see measurable quantity. surement procedure, or for assigning values to materials (ISO-Guide 30-2.1). random error of measurement; random error: reference population, Result of a measurement minus the reference individual: mean that would result from an infinite Reference individuals are persons number of measurements of the same selected by inclusion and exclusion cri- measurand carried out under repeata- teria from a healthy population to form bility conditions. the reference populations from which reference values are obtained for com- reagent: parison with an individual having a Substance employed to produce a specific disease. The reference popula- chemical reaction in order to measure tion should be as similar as possible to quantities, pertaining to other sub- the persons tested except for the dis- stances or convert one substance into ease being investigated. another. repeatability of results of receptacle: measurements; repeatability: Vessel, wether evacuated or not, in- Closeness of agreement between the tended to contain a specimen, together results of successive measurements of with any receptacle accessory and ad- the same measurand carried out under ditive, with closure in place. repeatability conditions. Receptacle accessory is a component inside the receptacle which is intend- sample: ed to assist in the collection, or mix- One or more parts taken from a system ing, or separation, of the specimen and intended to provide information on (89). the system, often to serve as a basis for a decision on the system or its production.

100 Glossary

A portion taken from a system is traceability: sometimes called a ”specimen”. Ability to trace the history, application It may be useful to distinguish be- or location of that which is under con- tween ”primary sample” (taken from sideration (CEN-ISO 9000: 2000). the original system), ”laboratory sam- Property of the result of a measurement ple” (as received by the laboratory), or the value of a standard whereby it and ”analytical sample” from which can be related to stated references, the ”analytical portion” is taken. usually national or international stan- See also analytical sample, patient dards, through an unbroken chain of sample (specimen). comparisons all having stated uncer- sampling: tainties (VIM; 1993, 6.10). Process of drawing or constituting sam- turn-around-time: ples, usually qualified by a description of Time interval between collection of pri- the sampling procedure (ISO 3534-2). mary sample (patient sample) and re- sampling procedure: port to the ordering health service, or Time interval between receive of re- Operational requirements and/or in- quest and report to the requesting structions relating to the use of parti- health service (90). cular sampling plan, that is the planned procedure of selection, withdrawal, unspecificity: and preparation of one or more sam- Effects of sample components other ples from an inspection lot to yield than the analyte that by themselves pro- knowledge of the characteristics of the duce a signal of the measuring system. lot (ISO 3534-2). Note: In laboratory medicine, the unit of measurement; unit: inspection lot usually is a person. Particular measurable quantity, defined specimen: and adopted by convention, with Biological material which is obtained which other measurable quantities of in order to detect properties or to mea- the same kind are compared in order sure one or more quantities (89). to express their magnitudes relative to that quantity. stability: Ability of a system, when kept under value of a measurable quantity; specificed conditions, to maintain a value: stated property value within specified Magnitude of a measurable quantity limits for a specified period of time. generally expressed as a unit of measurement multiplied by a number. sterility: Sterility is the absence of living organ- venipuncture: isms. All steps involved in obtaining an appropriately identified blood spe- systematic error of measurement; cimen from a person’s vein. systematic error: Means that would result from an infinite number of measurements of the same measurand carried out under repeatability conditions minus a true value of the measurand.

101 Index

A atrial natriuretic peptide (ANP) 13, 18 cerebrospinal fluid (CSF) cytology 27 autoantibodies 79 ceruloplasmin 7, 82 acetate 13 chemical waste 48– 49 acetoacetate 8 chlorazepam 69 N-acetyl-procainamide (NAPA) 70 B chloride 19, 29, 57, 66, 77, 80 acid citrate dextrose (ACD) 35, 61, 62 bacteria 72 cholecalciferol (25-OH) 14 acid phosphatase 7, 32, 42 bacteriological testing 72 – 75 cholecystokinin-pancreozymin 59 ACTH (see corticotropin) bar-code 24–25 cholesterol 7– 8, 12–13, 16–17, 18, 80 activated partial thromboplastin time bed rest 18 cholinesterase 7, 9, 32, 82 (APTT) 17, 52– 53 bicarbonate 8, 13, 57 chronobiological influence 14 –15 acute phase proteins 7 biguanides 82 chylomicronemia 15 addictive drugs 13 bilirubin 6 –9, 13, 16, 19, 32, 37, chylomicrons 57, 76 additives 34 –35 40, 81, 82 cigarettes 12 adenylate kinase 81 biohazard 38 circadian rhythm 14 –15 ADH (see vasopressin) biological halflife 69 cis-platinum 82 adrenaline (see epinephrine) biological influences 5, 13, 82 citrate 16, 30, 34, 52–53, 55 age 6 biopsies 16 citrate buffered 52, 60 air temperature 10 blank 80 citric acid/theophilline/adenosine/ alanine aminotransferase (ALT) 8 – 9, blood cell lysis 62 dipyridanole (CTAD) 55 13, 19, 36 – 37, 80, 82 blood cells 54 –55, 60, 62– 65 clinical chemistry 56–57 albumin 7–9, 17, 18 –19, 27, 30, blood collection 60 – 61 clonazepan 70 32, 37, 83 blood culture 21 clot activators 20 alcohol (ethanol) 13, blood gas analysis 21, 23 clot formation 32 aldosterone 12–13, 14–15, 17, 18 blood gases 21, 66 – 67 clotting factors 7, 32 alkaline phosphatase 6–7, 9, 18–19, blood grouping 16, 50 – 51 caffeine 12 32, 37, 76 blood smear 37, 55 coagulation 52– 53 allopurinol 82 blood transfusion 50 –51 cocaine 69 altitude 11 body temperature 15 coffee 12 amikacin 70 borate serine EDTA 36 cold agglutination 78 amino acids 7 bronchoalveolar lavage (BAL) 73 color codes 34 – 35 amiodaron 70 competition for binding sites 83 amitriptiline 71 complement activation 50, 59 ammonia 7, 9, 32– 33, 36 C complement components 59 ammonia excretion 29 cadmium 12 condensation 40 amphetamine 13 calcium (free or total) 8 –9, 18 –19, contamination 16 –17, 69 amylase 6, 13, 16, 79, 80 28 – 30, 37, 40 contamination control in molecular analyte absorption to the vessel wall cannabis 13 biology 64– 65 28 capillary sampling 22–23, 66, 84 contamination with the anticoagulant analytical time 85 – 86 carbamazepine 70 21 angiotensin 17 carbohydrate deficient transferins 13 conveyer belt system 45 carbondioxide (pCO ) 13, 66 – 67 angiotensin converting enzyme 12 2 copper 7, 12, 78 antibodies 37, 78 – 79 carboxyhemoglobin (CO-Hb) 12 corrosive chemicals 48– 49 anticoagulant 21, 32, Annex carcino embrionic antigen (CEA) 12 corticotropin (ACTH) 9, 15, 58 β antiphospholipid antibodies 79 -carotinoids 12 cortisol 9, 13, 14–15, 17, 58, 82 apolipoprotein AI 7, 18, 41 cartesian robot 45 cotinine 12 apolipoprotein B 8, 18, 41 casts 28 CO2 57, 66 aprotinin 53, 59 catecholamines 17, 30, 59 C-peptide 58, 59 33 arterial puncture 20–21 cation interference C-reactive protein (CRP) 11, 16 61 arterialization of the puncture site 23 cellular analysis creatine kinase 6–10, 19, 32, 40, 60– 61 aspartate aminotransferase (AST) cell separation tube 77, 80 33, 42–43, 76, 84, Annex 7– 9, 13, 32– 33, 18–19, 37, 80 centrifugation creatinine 2–3, 7–9, 16, 19, 28, 82 “atraumatic“ pencil shaped needle cephalotin 29– 30, 37, 57, 77, 80 26 cerebrospinal fluid (CSF) 26 –27, 69

102 Index Samples: From the Patient to the Laboratory. W. G. Guder, S. Narayanan, H. Wisser, and B. Zawata Copyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-30981-8 creatinine clearance 7, 11 EDTA-aprotinin mixture 53 G cristals in urine 30 EDTA dependent antibodies 78 gas diffusion 40 cryoglobulins 78 EDTA plasma 58 gas exchange 67 cyclic AMP 12 EGTA 59 gastrin 58 cyclophospamid 82 effect of light 40 gastro-inestinal tract surgery 16 ciclosporine A (CsA) 69, 71 electrolytes 56, 57, 66 gel barrier 60 – 61 cylindrical robot 45 elimination half-life of drugs 70 –71 gender 7 cytotoxicity 82 endorphin 58 gene rearrangements 62 β -endorphin 58 genes 62– 65 D enzyme induction 82 gentamicin 70, 82 eosinophils 15 γ date of birth 24 -globulin 16, 33 epinephrine 9, 12–13, 15, 18 D-dimer 53 glas capillaries 67 ergometry 16 deproteinization 56 glas syringes 67, 69 European standard on sample trans- desipramine 71 glomerular filtration rate 7 portation 39 detergents 28, 63 glucagon 8–9, 59 erythocyte sedimetation rate 7 dextran 16 glucose 2– 3, 7–9, 12–13, 16 –17, erythocytes 7, 18–19, 37, 78 diabetes mellitus 2 19, 21, 30, 36, 40, 57, 77 estriol 11 diagnostic and therapeutic interven- glucose tolerance 8, 14 estradiol 13 tion 16 glutamate dehydrogenase 13 ethanol 13 diagnostic and therapeutic proce- glutamine hydrolysis 32 ethosuximide 70 γ dures 14 –15, 16–17 -glutamyltransferase 7, 9, 13, 19, evaporation 40 diet 8, 84 32, 37, 80, 82 exercise 9–10, 14, 16, 19 diethylpyrocarbonate (DEPC) 64 glutathione 59 explosive chemicals and waste digitoxin 68, 71 glycerol 8, 12 48 – 49 digoxin 68, 71, 82 glycolysis 40, 67 extraction 84 digoxin-like immunoreactive glycolytic inhibitors 21, 34 – 35 substances 69 glycoproteins IIIA, IIb 79 disopyramide 70 gold standard 5, 87 granulocyte lysis 80 direct bilirubin 32 F direct potentiometry 56 granulocytes 6, 61 factor VII 53 disposal 46 – 49 granulocyte viablity 61 factor VIII 53 disposal of chemicals 48 – 49 growth hormone 58 factor XI 53 disposal of needles 46, 48 guanidinium isothiocyanate 63 factor XII 53 disposal of specim 47– 48 gut glucagon 58 femoral artery 21 disposal of sharp objects 46 – 47 ferritin 7 distribution of sample 42– 43, 84 fibrin degradation products (FDP) 53 diurnal changes 58 fibrin monomers 17, 41 H diurnal variation 14–15 fibrinogen 12, 16 –17, 32– 33, 53 haematin 62 DNA (desoxyribonuclein acid) fibrinolysis 53 haematocrit 9, 11, 12, 18, 37, 52, 62– 65 FICOLL-HYPAQUE 60 – 61 56 documentation 85 – 86 finger puncture 22–23 haematology 54 – 55 dopamine 32 first morning urine 28 haemoglobin 6 –7, 11, 15, 17, drinking 8 flammable waste 48 – 49 18 –19, 37, 80 – 81 drug colour chart 83 flow cytometry applications 61 haemoglobin A1c 3– 4, 82 drug effects and interferences 82–83 fluoride 26, 34 – 35 haemolysis 33, 50, 80 – 81, Annex drug monitoring 15, 31, 68 –71 folate 13 haemostasiology 52– 53 drugs 68 –71, 82 – 83 folic acid 40 hazardous waste 48– 49 “dry chemistry“ 56 follitropin (FSH) 58 HDL-cholesterol 6 –7, 12, 18, 80 duration of storage 36 food depriviation 8 heavy metals 12 food intake 8 heparin 34, 58 – 59, 60 – 61, 62, 66, E free fatty acids 8, 13 68 edema formation 18 free hemoglobin 81 heparin interference 33, 62 EDTA (ethylenediaminetetraacetic- freezing of blood 69 heroin 13 acid) 2, 34, 50, 54 – 55, 58 – 59, fructose 16 heterophilic antibodies 79 61, 62, 68 – 69 fungi 73 – 74 high fat diet 8

Index 103 human leucocyte antigen (HLA lactate 8, 10, 13, 17, 19, 21, 27, mixing bar (“flea“) 66 typing) 60 32, 40, 67, 82 mixing of deep-frozen samples 41 hydrochloric acid (HCL) 30 lactate dehydrogenase (LDH) 19, mixing of specimen 20, 67 5-hydroxy indolacetic acid 30 32– 33, 37, 42, 43, 80 molecular biology 62– 65 hydroxyproline in urine 7 LDL-cholesterol 6 –7, 12, 18, 41 mononuclear cells 61 hydroxybutyrate 8 lead 12 monocytes 6, 12, 60– 61 hydrolysis of glutamine 32 leucocytes 18 –19, 27, 37, 78 – 79 morphine 13 hyperlipidemia 76 – 77 leupeptin 59 murine monoclonal antibodies 79 hypotonic lysis 61 lidocaine 70 muscle mass 7, 10 hypoxia 10 light effects 40 mycological testing 73 – 74 lipase 13 mycobacteria 29 lipemia 76 – 77, Annex myoglobin 7 I lipemic sample 76 – 77, Annex icteric sample Annex lipids 57 identification of patient 20, 50, 84 lipoprotein(a) 12 N + + identity of samples 24 –25, 41 lipoprotein electrophoresis 41 Na , K -ATPase 57 imipramine 71 lipoprotein X 41 nafamostate mesylate 59 immune hematology 50 –51 lithium 32, 71 needle disposal 46 – 48 immunoassays 59 long term starvation 8 needle reshielding 47 immunocytology 27 lutropin (LH) 58 netilmicin 70 immunoglobulin A 18 lymphocyte separation 60– 61 neuron specific enolase 32 immunoglobulin G (IgG) 7, 18, 27, 41 lymphocytes 6, 12, 60, 78 neurotensin 13, 58 immunoglobulin M 18 lysis of red blood cells 62 neutrophils 12 indirect sample identification 25 lysis of the cells 32 neutrophil morphology 54 infectious substance label 38 nicotine 12 influence of temperature on serum nitrazepam 69 analytes 36 M non-ionic detergents 63 norepinephrine 9, 13, 15, 18 information flow 24 macroamylase 79 nortryptiline 71 infusion site 17 macro creatinine kinase (CK) 79 novobiocin 82 infusions 16 –17 macroenzymes 79 injections 16 magnesium 29–30 inorganic phosphate 8 –9, 16, 19, magnesium phosphate 41 O 29– 30, 32, 36, 42 mailing of blood 36–37, 38 –39 oligoclonal protein bands 27 insulin 8–9, 13, 16–17, 58 malnutrition 8 operations 16 interference 33, 76 – 77, 80 – 81 marathon race 9 optical interference 80 interference factors 5 material safety data sheets (MSDS) oral contraceptives 82 iodoacetate 35 49 oral glucose tolerance test 16, 22 ionising radiation 16 Mean cellular (corpuscalar) volume order of collection 21 ionized calcium 66 – 67 (MCV) 12–13, 37, 54, 78 osmolality 18, 30 ion release from blood cells 67 meal 8, 15 osteocalcin 13 iron 7, 15, 32, 82 menstrual cycle 14 overfilling tubes 20 isometric exercise 9 mental stress 16 –17 ovulation 14 isopropanol 22 metabisulfite 59 oxalate 30, 68 isotonic exercise 9 metabolic acidosis 13 oxygen (pO2) 67 metabolism of blood cells 67 J method-dependent interference 33, 81 methotrexate 71 P jointed robot 45 microbiology testing 72 – 75 pancreatic peptide 58 microbiology in CSF 26 –27, 73 pancreatic polypeptide 13 K micro blood collection sets (disposal) parasites 74 – 75 47– 48 parasitological testing 74 – 75 ketone bodies 8–10 microcollection technique 23 parathyroid hormon related protein microcollection tubes 42– 43 (PTH-RP) 59 L mid-stream urine 29 patient preparation 84 labeling 80 mitochondrial DNA 62 patient’s criteria of quality 85 laboratory computer 24–25 mixing 55, 67, 84 pepsinogen-1 58

104 Index pepstatin 59 protein electrophoresis 33 sample sorter 44 pH 8, 21, 40, 67 proteolytic enzyme inhibitor 58 sample treatment 84 pharmacological effect 82 prothrombin time (PT) 17, 52- 53, 82 sampling 14 –15, 26 – 27, 52, 58, 67, phenobarbitol 70 pseudoagglutination 16 68 –71 phenol 63 pseudoleucocytosis 78 sampling from catheters 17, 21 phenprocoumon 82 pseudothrombocytopenia 55, 79 satellism of platelets 55 D-phenylalanine-proline-arginine- pseudothrombocytosis 78 scalp artery 21 chlormethylketone 53 proteolytic enzyme inhibitor 58 – 59 scheduling infusions and blood sam- phenytoin 70, 82 – 83 pyridoxal phosphate 12 pling 17 pH value in blood 16, 21, 66 – 67 pyruvate 8, 19 seasonal influences 14 phlebotomy 20 –21 pyruvate kinase 9 second morning urine 28 phosphate, phosphorus 8–9, 15, 16, secretin 58 –59 22, 30, 32 selection of vein 84 phosphodiesterase 12 Q selenium 12 plantar surface of the heel 22 quality assurance 84 – 87 serotonin 32 plasma 16, 32– 33, 57 quality managers 86 serum 16, 32– 33 plasma-serum differences 32– 33 quality manual 86 serum osmolality 11 plasma water 56 quality of the sample 85, Annex serum-plasma differences 32– 33 plastic syringes 67, 69 quality of timing 85 site for sampling blood 20, 22– 23 plastic tubes 66 quinidine 70 skin puncture 20, 22– 23 plasticizers 69 sleep 14 platelet contamination 43 R smoking 12 platelet count 32, 55 sodium 8 –9, 13, 15, 19, 30, 32, 37, race 6–7 platelet derived growth factor 55 57, 66, 77, 80 radial artery 21 platelet factor IV (PF4) 55 sodium azide 30 ratio of anticoagulant to blood 52, 54 platelet free plasma 33 sodiumborate serine EDTA 36 red cell mass 12 platelet lysis 80 sodium carbonate 30 refrigeration 58 platelet poor plasma 33 sodium dodecylsulfate (SDS) 63 renin 11, 13, 15, 17, 18, 58 platelet rich plasma 33 somatotropin (growth hormone) 9, reptilase time 16 –17 platelet satellism 55 15, 17 request 84 platelets 32–33, 43, 54 –55, 60, 61, somatostatin 58 – 59 request form 24, 84 78 soybean trypsin inhibitor 53 reshielding of needles 47 pneumatic tube delivery system 36 specimen collection 28 – 31, 52, 55, 66 restriction fragment lenght polymor- polyagglutination 50 – 51 specimen collection bags 28 phism (RFLP) 62– 64 polymerase chain reaction (PCR) 62, specimen processing 42 – 43 reticulocytes 7 65 spironolactone 82 retinol binding protein 8 porpyrins 30, 40 sputum tube 74 – 75 rethawing effects 40 –41 position of patient 20 stability of the constituents in the sam- ribonuclease (RNAses) contamination post-centrifugal coagulation 33 ple 30, 40 – 41, 55, 69 64 posture 18, 58, 84 stabilising RNA 64 rimixing after storage 41, 84 potassium 2– 4, 8 – 9, 13, 15, standard meal 8 RNA (ribonucleic acid) 62– 64 16 –17, 19, 22, 29– 30, 32– 33, starvation 8, 15, 29 robotics 44 – 45 36 – 37, 42, 57, 66– 67, 80 – 81 steady-state of drugs 70 –71 rubber closures 69 pO 67 stool tube 73 – 75 2 running 9–10 preanalytical times 85 – 86 storage of samples 27, 30, 40– 41, pre-ovulatory increase 15 55, 57– 58, 64, 67, 69, 84, Annex pregnancy 7 S storage temperature 30, 36, 51 preservation of cells 35 safety aspects 46 – 49 streptokinase 53 primidone 70 safety flow lancet 22 streptomycin 70 procainamide 70 safety labels 49 subsamples 24 proinsulin 58 salicylates 78 substance P 58 prolactin 7, 12–13, 15, 17 saliva collection 31, 69 supine position 18 protease inhibitor 59 sample handling 43, 55 surgical procedures 9, 16 protein: see total protein sample identification 24 – 25 sustained-release preparations 70 protein binding of proteins 70 –71, 83 sample inhomogeneity 40, 72 swap specimen and tube 73 – 75 protein C 78 sample preparation 64, 67 swimming 9

Index 105 T tourniquet 19, 20, 66, 84 urine pH 30 tacrolimus 71 tourniquet application time 19 urine preservation 30 Taq DNA polymerase 62 toxic chemicals 48 – 49 urine sediment 41 temperature (body) 15 trace elements 57 urine volume 7, 15 temperature control 84 training status 9–10, 11 urobilinogen 30 temperature during transport 36 –37 transcortin 78 urokinase 53 testosterone 15 transferrin 32 thawing samples 40 – 41 transfusions 16 –17 V transport of diagnostic specimen 27, theophylline 68, 71 valproic acid 70, 83 36 – 37, 38 – 39, 53, 55, 58, 64, therapeutic drug monitoring (TDM) vanillylmandelic acid (VMA) 13 67, 69, 84 68 –71 vancomycin 70 transport of analytes 55 thiocyanate 12 vasoactive intestinal peptide 58 tricyclic antidepressants 69 thrombin 53 vasopressin 13, 17 triglycerides 7– 8, 13, 18 –19, 32, thrombin time 16 –17 venipuncture 20 – 21 β 56, 57, 76 – 77, 80 -thromboglobulin 55 very low density lipoproteins (VLDL) triiodothyronine (T3) 8, 32, 79 thrombocytosis 67 9, 76 thrombocytolysis 32 – 33 thromboxane A2 55 viruses 75 thrombocytosis 67 thymol 30 volume displacement effect 56 tube and sample disposal 47 thyroid-stimulating hormone (TSH) volume errors 33 turbidity 15, 76 – 77 13, 15, 17, 32 volume of blood collected 21 turbid samples 76 – 77 thyroxine 7, 9, 13, 15, 18, 79, 82 volume shift 10, 18–19 turn around time (TAT) 44, 85 – 86 thyroxine-binding globulin 82 von Willebrand factor 16 time after drug 14–15 two-dimensional bar code 25 time after last meal 14–15 two-dimensional dotcode 25 time after last sample 14–15 W time dependent changes 14 U warfarin 82 water displacement 77 time of sampling 14–15, 24 ultracentrifugation 77 white blood cell differential count 54 time of the day 14–15 umbilical artery 21 whole blood 57, 67 time saving 33 upright position 18 whole blood lysis 61 timing 14–15, 24, 58, 68, 84 – 85 urea 7–9, 16, 19, 29 – 30 workflow 44, 45 timing of storage 84, Annex uric acid 7–11, 13, 16, 19, 22, 30, tobacco smoke 12 41, 73, 78 tobramycin 70 urinary creatinine 10 –11 total bilirubin 32 urinary excretion of calcium 18, 28 total protein 7, 16, 18 –19, 30, 32, urine 28 – 30, 69 77, 82 urine containers 28, 84

106 Index Competence in laboratory procedures

G.I.T. “Imaging and Screening – Trends in Management & The well-established, Microscopy” offers a Drug Discovery focuses Krankenhaus is a source pan-European bi-monthly qualified international on the latest trends and of information for de- journal, EUROPEAN forum for the presentation developments in the cisionmakers in hospitals HOSPITAL, is aimed at of products, services field of active substance and is also aimed at decisionmakers in and company. This inter- research and trends in representatives of health healthcare organisations national publication is drug discovery. It covers insurance organisations, throughout the EU and published in English and all aspects of research professional associati- those in greater Europe. is distributed to decision- as well as strategies of ons and industry, amon- Highly varied editorial makers and opinion pharmaceutical com- gst others. “Networ- coverage includes news leaders in industry, panies and start-up king” is the motto for the of clinical & technolo- research and at universi- companies. This editorial future and the construc- gical advances, admini- ties in Europe. mix means that this tive collaboration of all stration and healthcare publication is relevant professional groups in politics, financial strate- ”Imaging & Microscopy” reading for all those the hospital setting is be- gies, exciting innovati- provides up-to-the-minute concerned with drug coming more and more ons, plus interviews with coverage of applications discovery. Internet important. For this rea- scientists working in and products in the and/or E-mail offer son, in addition to its re- R & D, as well as people areas of image proces- readers the possibility gular columns, each is- on the cutting edge of sing, microscopy and of direct contact without sue of M & K focus on healthcare, manufactu- other modern imaging delay. Screening – special interdisciplinary ring, and policy making. processes. The methods Trends in Drug Discovery topics, thereby offering EUROPEAN HOSPITAL and instruments invol- is published in English. a platform for dialogue, is published in English. ved, such as sample highlighting trends, and preparation and sample making visions known. handling, are presented The special “Pain” and in a practical, easy-to- “Modern Cancer Thera- understand manner in py” and the magazine an attractive layout. “MedAmbiente” round of this series of journals. Published in German.

Roesslerstrasse 90 · 64293 Darmstadt · Germany · Tel. (+49) 61 51/80 90-0 · Fax (+49) 61 51/80 90-146 www.gitverlag.com Samples: From the Patient to the Laboratory. W. G. Guder, S. Narayanan, H. Wisser, and B. Zawata Copyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-30981-8