samples from the patient to the laboratory

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

3rd, revised edition 2003

Samples: From the Patient to the Laboratory. W. G. Guder, S. Narayanan, H. Wisser, and B. ZawataCopyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 978-3-527-30981-8

1st Edition, 19962nd Edition, 20013rd 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 itemsmay inadvertently be inaccurate.

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Bibliographic information published byDie Deutsche BibliothekDie Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie;detailed bibliographic data is available in the Internet at <http://dnb.ddb.de>.

© 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

All rights reserved (including those of translation into other languages). No partof this book may be reproduced in any form – by photoprinting, microfilm, or anyother means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used inthis book, even when not specifically marked as such, are not to be consideredunprotected by law.

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Composition 4t Matthes + Traut Werbeagentur GmbH, DarmstadtPrinting Druckhaus Darmstadt GmbH, DarmstadtBookbinding Litges & Dopf, Buchbinderei GmbH, HeppenheimISBN 3-527-30981-0

III

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 contributionof these factors on laboratory results. They agreed to summarize their knowledge in a short and understandable form aiming to increase the awareness of these factorsamong all professions involved in the preanalytical phase of the laboratory diagnos-tic 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 meetingof the authors together with the publisher, the manuscripts were completed by theauthors in a short time with the help of many collaborators and colleagues. Theauthors 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 wouldlike 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 availableand 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 closecollaboration with all contributors.

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

Walter G. Guder Sheshadri Narayanan Hermann Wisser Bernd Zawta

May 2003

V

Contents

Samples: From the Patient to the LaboratoryThe 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 .......................................... 02The 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

Contents

VI

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

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

VII

Contents

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

IX

Foreword to the First Edition

aboratory tests generally provide a more sensitive indicator of the state of apatient‘s health than the patient‘s account of how he or she feels. This hasprompted an increasing emphasis on laboratory tests in the diagnosis and ma-

nagement of the patient‘s disease. Major decisions about the management of a patientare being made on small changes in laboratory data. Thus, a decision to change thedose of a patient‘s drug is often made on its plasma concentration.

Laboratories have long been aware that many non-disease factors may affect clinicallaboratory test values. These include the potential effect of drugs, either through aneffect on the physiological function of various organs, or an interference with ananalytical method.

Whereas the laboratorian may be aware of the possibility of an analytical inter-ference, clinicians are largely unaware of these effects and the available resourcesto help them interpret test values correctly. When this information is not given withthe result, clinicians may misinterpret test values and take an inappropriate actionwith their patients.

Clinical decisions based on laboratory test values are correctly made only when theconditions under which blood or other specimens are properly identified and stand-ardized, or when the lack of standardization is recognized and allowances aremade for some lack of comparability with previous test values. While laboratoriansare aware of the concepts of intra- and interindividual variation as they affect labo-ratory data, many colleagues are unfamiliar with all but the most obvious causes ofdifferences in test values, such as gender and age.

An understanding of intraindividual variation of test values is important if appropriateclinical decisions are to be made when serial data are being followed. The new con-cepts of critical differences or reference changes are now important. For proper in-terpretation of the typically small differences between laboratory data obtained onsuccessive specimens from patients, the variables affecting the test values need to bestandardized 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 preana-lytical variables. It is thus particularly timely for this book to be published. The aut-hors hope to reach a broader audience than the laboratorians who are probablyquite familiar with many of the factors affecting test results. Since 1956, when RogerWilliams published his pioneering studies on the differences between people in abook entitled “Biochemical Individuality“, physiologists have been concerned withthe differences between people. Now that we have a broader understanding of thegenetic influence on human physiology and behavior and a greater need to extractmore information from small changes in laboratory data, the publication of a newbook concerned with preanaytical variables which contribute to intra- and inter-individual variability is both timely and welcome. This book is intended not just forlaboratorians but also for physicians, nurses and everyone involved in the chain ofevents 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.

L

Dream and reality

02

A new patient with diabetesmellitus is encountered

Mrs. Haseltine is a 56-year-old ladywho lives in a remote area. She consultsher nearby practitioner and reports thatover the last two weeks she has urinat-ed more frequently than usual. Also,her body weight has decreased, al-though she “drinks more soft drinks thanever before“. The practitioner finds apositive dipstick result for glucose inher urine. Using a glucometer, hemeasures glucose from fingertip bloodobtained by pricking with a fine lancet.The first drop of blood is washed awaywith a swab of gauze. In the followingdrop, glucose is measured by the meter,a process that takes about 30 seconds.The result is 280 mg/dL (15.56 mmol/L),far above the upper limit of the normalrange. Mrs. Haseltine is informed thatshe may have diabetes mellitus and isreferred to a diabetologist the next day.

The right sample for the right testat the right time

The diabetologist confirms the resultobtained by the practitioner using acapillary blood sample taken 1 hourafter breakfast.

Two blood samples are drawn from thepatient the following morning (after shehas fasted for 12 hours), from the ante-cubital vein into closed tubes, one, witha lavender-colored stopper, containingEDTA, the other, with a green cap,containing heparin. Mrs. Haseltine isinformed that she has type II diabetesmellitus and will have to be placed ona diet in order to treat her disease. Sheis asked to phone the next day toobtain information on her laboratoryresults and for further advice.

In the meantime, the heparin bloodsample has been centrifuged to sepa-rate plasma from the cellular elements.Both tubes are sent to the laboratory bycourier in a container especially de-signed to keep samples at constant tem-perature. The laboratory receives thesamples together with the patient’s dataand requests for determinations: glycat-ed haemoglobin and blood cell countsfrom the EDTA blood; potassium andcreatinine from the plasma, which hasbeen separated from blood cells, in theclosed heparin tube.

The laboratory technician identifies allthe samples by comparing the nameand bar code number with those on therequest sheet. He then enters the requestinto the lab computer. The samples areput into bar code-reading analyzers foridentification and performance of therequested tests. A subsample is takenfrom the EDTA blood – after slowlymixing it for 3 min on a roller mixer –for the determination of haemoglobinA1c by chromatography. The laborato-ry report, shown in Tab. 1- , is sent tothe diabetologist the next morning.

1

Fig. 1-1

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

03

An introductory case

Tab. 1- Laboratory report

Haseltine, Elsa July 13, 1994 10 a.m.Patient ReferenceResult Interval

Haemoglobin A1C 8.5% 2.5–6.0Haemoglobin 14.4 g/dL 12–15Potassium 3.6 mmol/L 3.5–4.5Creatinine 1.0 mg/dL Below 1.1

Mrs. Haseltine is taken into the neigh-boring county hospital together with aletter from the diabetologist informingthe clinician about all tests performedand the results obtained. After twoweeks of treatment, Mrs. Haseltine haslearned to control her blood sugar us-ing a small glucometer. No furthertreatment was needed for the next fewyears.

This is what might happen in reality

Mrs. Haseltine goes to the practitionerwith the same symptoms for the samecondition. In contrast to the positive urinedipstick result for glucose, the bloodsugar is nearly normal (120 mg/dL).The practitioner, to play-it-safe, againrefers the patient to a diabetologist.

One week later, Mrs. Haseltine is calledin for a glucose tolerance test. The onlyadvice she is given is to fast the nightbefore the test. Mrs. Haseltine wakes up

1 late, however, and misses her morningappointment. She arrives at the doctor’soffice at noon, having had a snack onthe way. She is stressed when the nurseoffers her a glucose-containing drinkafter taking a “fasting“ blood specimen.

She feels nauseated while slowly consum-ing the drink. Whilst waiting for the nurse,she decides not to drink it all and emptiesthe remaining drink down the bathroomsink. Of course, she doesn’t report thisincident to the nurse when she returns totake a capillary blood sample at one andtwo hours after the first sample.

When the results are shown to the doctor(Tab. 1- ), he realizes that the glucoseconcentrations after the first and se-cond hour are not that much different.The diabetologist, unable to arrive at adiagnosis, asks the patient to report thefollowing day at which time two venousblood samples are collected, one with a lavender-colored stopper and the oth-er with a green cap. The tubes are sentto a private laboratory by car. Nextday, the results shown in Tab. 1- arereceived by telefax together with thereference values for each test. The glu-cose value is now normal, potassium el-evated and haemoglobin A1c, an indi-cator of mean blood glucose, elevatedto diabetic levels. The diabetologist,concerned by the high potassium level,refers the patient to a clinic. This institu-tion diagnoses that the patient has typeII diabetes mellitus, based on their la-boratory results.

Tab. 1-Results in doctors office: glucose tolerance testHaseltine, Elsa July 12, 1994 – 2 p.m.

Fasting Glucose 160 mg/dL1-Hour Glucose 110 mg/dL2-Hour Glucose 120 mg/dL

2

3

2

Fig. 1-2

Dream and reality

04

Tab. 1- Report from private laboratoryHaseltine, Elsa – July 13,1994 3 p.m.

Units Patient's Result Reference Range

HbA1c % 8.5 3.5 – 6.0Haemoglobin g/dL 13.5 12 – 15Potassium mmol/L 5.8 3.5 – 4.5Creatinine mg/dL 1.0 Below 1.1Glucose mg/dL 105 70 – 110

What happened to Mrs. Haseltine's samples?

Undoubtedly, Mrs. Haseltine was in adiabetic state. Why was the fastingblood sugar nearly normal?

Answer: Fasting may result in nearnormal values in type II diabetics. Inthis case, the nurse took the first dropof blood from a fingerprick after“milking“ the finger to obtain sufficientblood.

Why was the result of the glucosetolerance test inconclusive?

Answer: The first result was related topatient stress, which leads to increasedamounts of glucose being releasedfrom liver glycogen stores. Moreover,Mrs. Haseltine had a snack on her wayto the doctor because she was hungry.

3

She did not report this to the doctor orthe nurse, because she wasn’t aware ofthe possible influence of this snack. Forthe same reason, she did not report notconsuming all of the glucose drink,which had led to a decrease ratherthan an increase of blood glucose afterone hour. The “increase“ at the secondhour may have been due either tomethod variation or to a reactiveincrease brought about by metabolicreactions in the late afternoon. Normally,a glucose tolerance test is performed inthe morning, the reference values be-ing valid only for the morning. It shouldbe carried out under standard condi-tions, as recommended by nationaland international expert panels.

Why was potassium elevated andglucose normal in the venousspecimen?

Answer: The sample was transported incontact with the cells for over two hoursin a non- air conditioned car on a hotday. This caused the blood cells tometabolize glucose and release potas-sium, the concentration of which isapproximately 40 times higher in cellsthan in plasma. This in-vitro influencemakes unstabilized blood unsuitablefor glucose determination. Potassium canbe reliably measured only if plasma ispromptly separated from the cells.

All these errors could have beenprevented had the preanalytical phasebeen strictly controlled. Mrs. Haseltinewould have been diagnosed earlierwith less stress, and fewer costs wouldhave been incurred.

Fig. 1-3

05

The importance of the preanalytical phase

This book is intended to increaseawareness of the importance of allsteps of the preanalytical phase, in-cluding patient preparation, sampling,transport and storage of patient samples.

In each chapter, covering two oppositepages, possible preanalytical variablesare explained with regard to mecha-nisms, effects and preventive actionsintended to prevent misinterpretation oflaboratory results. In the respectivechapters, warnings are given in redand recommendations in green. Likedisease mechanisms, biological influ-ences can change the concentration ofmeasured analytes in-vivo, whereas in-vitro changes have to be separatedinto changes undergone by the measuredanalyte and interference of the method used to measure the analyte.These definitions are important, be-cause only the latter can be avoidedby using a more specific method.The interested reader is referred tothe literature summarized onpages 88 – 95 as well as theglossary which defines all thespecial terms used in thisbook (p. 97). A detailed in-formation on preanalyticalvariables of all analytes to-gether with the recommen-dations on the choice of anti-

Optimaltreatment of patient and his samples is defined as the gold standard

coagulant, the optimalsample volume andthe stability of an-alytes in samplematrix is includedin the Annex: TheQuality of Diagnos-tic Samples.

Something unavoidable

06

Fig. 2-2Influence of race on

creatine kinase, amylase and

granulocytes in blood.P = pancreatic

isoenzymeS = salivary

isoenzyme

Intrinsic influences such as race, genderand age may influence target analyteconcentrations in clinical chemistry andhaematology. These variables are indi-vidual features of a subject and hencenot subject to change. Quite often,intrinsic and external factors are difficultto distinguish.

Age

Age may affect blood and urine ana-lyte concentrations after birth, duringadolescence or in old age (Fig. 2-1).Erythrocyte counts and hence haemo-globin are much higher in neonatescompared to adults. Within the first fewdays following birth, increased arterialoxygen provokes erythrocyte degrada-tion. The resulting increase in haemo-globin leads in turn to enhanced con-centrations of bilirubin. Since liverfunction (here in particular glucuroni-dation) is not fully established inneonates, increased concentrations ofbilirubin are observed.

Uric acid concentrations in neonates arein a range similar to adults. However,within days after birth, a significant de-crease is observed. Other examples ofage-dependence include alkaline phos-

phatase activity (AP) in serum (whichpeaks during the growth phase, mirror-ing bone osteoblast activity) and totaland LDL-cholesterol. In addition, age-dependent AP activity and LDL- andHDL-cholesterol in serum are influencedby gender. These gender differences inturn change as a function of age.

Race

Fig. 2-2 illustrates examples of analyteswhich are affected by race. Black Ameri-cans of both genders have significantlylower white blood cell counts comparedto whites. This difference is readilyexplained by a reduction in the numberof granulocytes. In contrast, haemoglo-bin, haematocrit and lymphocytecounts are almost identical in bothgroups (97). The monocyte count inwhites exceeds that of blacks (11). Asignificant difference in creatine kinase(CK) activity has been observed forboth genders in black and white peo-ple. This difference is not due to differ-ences in age, height or body weight(81). A substantial difference in amy-lase activity has been established be-tween West-Indians and native Britons.Based on the generally acceptedthreshold value, 50 percent of West In-dians had elevated amylase activities(219). Significant racial differences have

Fig. 2-1Age dependence of

various substrates andenzyme activity (8, 35).Alkaline phosphatase

was measured at 30 °C (86 °F)

200

400

600

800

140

200

200

300

160100

100

60

20

15 25 35 45 552 4 6 6 8 1012141618

1

2

3

4

5

birth

haemoglobin

uric acid

bilirubin

alkalinephosphatase

days years years

cholesterol

LDL-cholesterol

HDL-cholesterol

6

7

8

+

+

+

+

µmol

/L

g/L U/L mmol/L

granulocytesG/L

200

whi

te

hisp

anic

asia

n

blac

k

briti

shw

est-

indi

anas

ian

blac

kw

hite

creatine kinaseU/L

300

200

PS

α-amylaseU/L

4

3

PS

PS

++ +

+

▼

▼


07

Influences of age, gender, race and pregnancy

been reported for serum concentrationsof vitamin B12 (1.35 times higher con-centrations in black people) (183) andLp(a) (2 times higher concentrations inblacks compared to whites). It is note-worthy in this context that neither ather-osclerosis nor mortality is higher inblacks with high Lp(a) (75).

Gender

As with many macroscopic featuresand gender-specific hormone patterns,gender differences can likewise befound in clinical chemistry and haema-tology (Fig. 2-3). The gender differenceof serum iron concentrations disappearsin patients older than 65 years. Otherexamples of gender differences are CKand creatinine. The serum activity orconcentration depends on muscle masswhich is in general more pronounced inmales. Certain athletic activities whichlead to increased muscle mass mayblunt this difference (see p. 9–10).

Pregnancy

When interpreting laboratory resultsduring pregnancy, it is necessary totake into account the gestational weekat which each sample was taken.

During a healthy pregnancy, the meanplasma volume rises from about 2.6 L to3.9 L, with probably little change occur-ring in the first 10 weeks of gestation,and a subsequent progressive rise upto the 35th week, at which time the val-ues level off. Tab. 2- describes themechanism underlying the changes inplasma during pregnancy.

The urine volume may also increasephysiologically by up to 25% in the 3rd

trimester. There is a 50% physiologicalincrease in the glomerular filtration ratein the last trimester. The well-known

1

changes in hormone production andthe plasma concentrations of fertilityhormones during pregnancy are ac-companied by changes in various ana-lytes, e.g. thyroid hormones, metabo-lites (amino acids↑, urea↓), electrolytes(calcium↓, magnesium↓, iron↓, zinc↓,copper↑), proteins (especially acutephase proteins↑), and some diagnosti-cally important lipids (triglycerides↑,cholesterol↑), enzymes (alkaline phos-phatase↑, cholinesterase↑), factors of theplasma coagulation system and compo-nents of the fibrinolytic system. The se-dimentation rate is increased five-foldduring pregnancy. The concentrationchanges are caused by different mech-anisms as increased synthesis of trans-port proteins, increased metabolic turn-over rate or dilution.

Fig. 2-3Male – femaledifferences related tothe mean value offemales as given in (35)

triglyceridescreatine kinaseγ-glutamyltransferasebilirubinalanine aminotransferasecreatininemyoglobinuric acidureaammoniaaspartate aminotransferasehaemoglobinacid phosphataseerythrocytesamino acidsalkaline phosphatasecholinesteraseironglucoseLDL-cholesterolalbuminimmunoglobulin Gcholesteroltotal protein

reticulocytesapolipoprotein AIcopperprolactinHDL-cholesterol

1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.90.8 1.00.9

07

Changing habits

08

Diet

Diet and drinking are major factorsinfluencing a number of analytes inclinical chemistry. From a practicalpoint of view, one should distinguishacute effects from those observed over

a longer period. A critical question indaily routine is whether a standard mealaffects target analytes. Fig. 3-1 showsthe percentage change in differentanalyte concentrations as a function offood intake (37, 206). Effects of 5 per-cent and less may be neglected, sincethey are clinically irrelevant. Therefore,samples for these analytes do not re-quire strict food deprivation. The extentof food-induced alterations in analytes

depends on the composition of thefood and the elapsed time betweensampling and food intake. The serumconcentration of cholesterol and triglyc-erides are influenced by various factorsas food composition, physical activity,smoking, consumption of alcohol andcoffee (51). Elevated levels of ammo-nia, urea and uric acid are observedduring a high protein and nucleotidediet. The changes occurring after astandard carbohydrate meal (75 g) arediagnostically helpful in testing glucosetolerance. On the other hand malnutri-tion and starvation may alter analyteconcentrations in a clinically relevantfashion. Early indicators of low proteindiet are reduced serum concentrationsof prealbumin and retinol-binding pro-tein. Some alterations in clinical chemi-cal analytes induced by starvation over48 hours are summarized in Fig. 3-2.Metabolic acidosis with a decrease ofboth pH and bicarbonate results froman increase in organic acids, mainlythe ketone bodies (acetoacetic acid, 3-hydroxybutyric acid).

Starvation

Changes in analyte concentrations in-duced by long-term starvation (4 weeks)are shown in Fig. 3-3 at the end of thestarvation period in comparison to theinitial values. The concentrations of bloodcholesterol, triglycerides and urea arereduced. In contrast, creatinine and uricacid concentrations are elevated. Theincrease in uric acid concentration dur-ing starvation periods even requirestreatment. The latter is due to reducedclearance of uric acid as a result of ke-tonemia (44). It is readily apparent thatlong-term starvation is closely associ-ated with reduced energy expenditure;hence, as a result, T4 and, to a largerextent, T3 concentrations are reduced inserum. Besides such alterations, urinary

Fig. 3-1Change of the serumconcentration of dif-ferent analytes two

hours after a standardmeal (37, 206)

30x

10x

5x

1x

3-hydroxybutyrate*

acetoacetate*

48 h

Incr

ease

(x-fo

ld)

14 h

free fatty acidspyruvate*, lactate*glycerolglucagoninsulin

1.781.251.161.151.151.0551.0521.0271.0181.0181.0161.0041.0041.0001.0001.000

triglyceridesaspartate aminotransferasebilirubinglucosephospatealanine aminotransferasepotassiumuric acidtotal proteinalbumincalciumsodiumalkaline phosphatasecholesterolurealactate dehydrogenase

2.01.0 1.25 1.5 1.75Relative change in concentration after/befor meal

Fig. 3-2Variation of several

analytes after 40-48 hstarvation (113).

* Starting point after14 h starvation

▼

▼

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

excretion of several compounds is like-wise affected by long-term starvation.Urinary excretion of ammonia and cre-atinine is increased whereas that ofurea, calcium and phosphate is reduced(231). Changes in analyte concentra-tions brought about by long-term starva-tion are similar to those observed following surgical procedures or in patients with a catabolic status.

In measuring quantitative urinary excre-tion rates, excreted amounts per dayare preferable to those per liter in orderto eliminate variations in drinkinghabits and water excretion.

MechanismsChanges may be due either to anincrease in reabsorption of the measuredanalyte (triglycerides, glucose, aminoacids), intestinal or liver metabolism ofreabsorbed metabolites (VLDL, urea,ammonia) or regulatory changes dueto food intake or deprivation (uric acid,γ-glutamyltransferase, cholinesterase,thyroxine, retinol-binding protein, ketonebodies).

RecommendationIn order to avoid misinterpretation oflaboratory results, sampling after 12 hfasting and reduced activity is recom-mended as a standard procedure.

Exercise

Before considering the influence of ex-ercise on target analytes in clinicalchemistry, two types of exercise have tobe distinguished. First, static or isomet-ric exercise of brief duration and highintensity which utilizes the energy (ATPand creatine phosphate) already storedin muscle and, second, dynamic or iso-tonic exercise of lower intensity and

longer duration (e.g. running, swimming,cycling) which utilizes ATP produced byaerobic or anaerobic pathways. In ad-dition, the effect of physical trainingand muscle mass should be mentioned.Acute changes of analytes during exer-cise are due to volume shifts betweenthe intravasal and interstitial compart-ments, volume loss by sweating andchanges in hormone concentrations(e.g. increase in the concentrations ofephinephrine, norepinephrine, glucagon,somatotropin, cortisol, ACTH and de-creased concentrations of insulin) (4,177). These changes in hormone levelsmay in turn alter the leukocyte count tomore than 25 G/L as well as increasingglucose concentrations. Fig. 3-4 shows

Fig. 3-4Increase of variousanalyte concentrationsafter a marathon race.Blood was drawn oneday before and 45 minafter the race (203)

potassium

sodiumalkaline phosphatase

calcium

albumin

glucose

inorg. phosphate

uric acid

ureaaspartate aminotransferase

pyruvate kinase

creatine kinase

1x 2x 3x 4x

anal

ytes

0deviation (%)

20 40 60–20–40–60

sodiumpotassiumcalciumchlorideproteinalbuminglucoseuric acidureacreatininecholesteroltriglyceridesalkaline phosphatasealanine aminotransferaseaspartate aminotransferaseγ-glutamyltransferasehaemoglobinhaematocritweight loss

Fig. 3-3Change (%) of clinicalchemical analytes after4 weeks starvationand a daily supply of33 g protein, vitaminesand electrolytes(44, 232)

09

Changing habits

10

changes in analyte concentrationsinduced by marathon running (203). Theextent of change depends on a varietyof individual and/or environmental fac-tors (e.g. training status, air tempera-ture and intake of electrolyte- and car-bohydrate-containing liquids during theactual run).

The changes observed (e.g. increasedalbumin) can in part be attributed tothe above-mentioned volume shift fromintravasal to the interstitium or to loss ofvolume by sweating. The increased uricacid concentration in serum is a conse-quence of reduced urinary excretiondue to increased lactate concentrations.Hypoxia-mediated creatine kinase (CK)increase depends on the training statusand hence shows a high degree of indi-vidual variability. The less physically fitan individual is the more pronouncedthe increase in CK. Training increasesboth the number and the size of mito-chondria which is associated with in-

creased capacity of the oxidative en-zyme system. This effect in turn increasesthe capacity of the muscle to metabo-lize glucose, fatty acids and ketonebodies in aerobic pathways. As a conse-quence, mitochondrial CK-MB increasesto more than 8 percent of the total CKactivity without evidence of altered my-ocardial function. Well-trained individu-als have a higher percentage of totalactivity in terms of the CK-MB of skeletalmuscle compared to untrained persons.Several other analyte concentrationslikewise depend on muscle mass andtraining status. Thus, plasma creatinine,urinary creatinine and creatine excre-tion increase and lactate formation afterexercise decreases in trained com-pared to untrained athletes. Vigorousexercise may cause erythrocytes or oth-er blood cells to be excreted in urine.These exercise-induced changes, how-ever, usually disappear within a fewdays.

11

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

Altitude

Some blood constituents exhibit signi-ficant changes at high altitude com-pared to findings at sea level.

Significant increases with altitude areobserved, for example, for C-reactiveprotein (CRP) (up to 65% at 3600 m),β2-globulin in serum (up to 43% at5400 m), haematocrit and haemoglo-bin (up to 8% at 1400 m) and uric

acid. Adaptation to altitude takesweeks and return to sea level valuestakes days. A significant decrease invalues with increasing altitude is foundin the case of urinary creatinine, creati-nine clearance, estriol (up to 50% at4200 m) serum osmolality, plasmarenin and serum transferrin (239).

account for concentration changes bydirect or indirect effects. Decreased an-giotensin converting enzyme activity(ACE) in smokers is believed to resultfrom the destruction of lung endothelialcells with a subsequent reduction in the

release of ACE into the pulmonary cir-culation and/or enzyme inhibition (76).The extent of changes also depends onthe amount, kind (cigarettes, cigars,pipes) and technique of smoking (withor without inhalation). Moreover, smok-ing-induced changes are influenced byage and gender (204).Fig. 4-2 shows the concentrations ofcotinine, thiocyanate and carboxy-haemoglobin, used as markers for thequalitative and quantitative assessmentof smoking habits. Cotinine has theadvantage of having a longer half-life(20– 28 h) than nicotine, the parentcompound (12– 15 min) (189).

LDL-cholesterolcholesterolhaematocritMCVfibrinogencopperred cell masscadmiumleadmonocyteslymphocytesneutrophilsCEA

angiotensin converting enzymeprolactinβ-carotinoidspyridoxal phosphateseleniumHDL-cholesterol

(%)0 +10 +20+30+40+50+60-10-20-30-40

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

12

Fig. 4-2Effect of smoking ondifferent blood ana-

lytes caused by smokeconstituents (141, 239)

CaffeineCaffeine is found in many constituentsof food ingested daily. Despite its wide-spread use, the influence of caffeine onvarious analytes in clinical chemistryhas not been investigated in detail.Caffeine inhibits phosphodiesterase andhence cyclic AMP degradation. CyclicAMP in turn promotes glycogenolysis,thereby increasing blood glucose con-centrations. In addition, the glucoseconcentration increases due to gluco-neogenesis via epinephrine. Activationof triglyceride lipase leads to a three-fold increase of non-esterified fattyacids (141). Quantification of hormonesand drugs bound to albumin is hamper-ed by the fatty acid-induced displace-ment effect. Three hours after the intakeof 250 mg of caffeine, plasma reninactivity and catecholamine concentra-tions have been found to be elevated(175).Studies intended to investigate theseanalytes should take caffeine consump-tion into account.

Effects of smoking

Smoking leads to a number of acuteand chronic changes in analyte con-centrations, the chronic changes beingrather modest. Smoking increases theplasma/serum concentrations of fattyacids, epinephrine, free glycerol, al-dosterone and cortisol (239). Thesechanges occur within one hour of smok-ing 1-5 cigarettes. Alterations in analytesinduced by chronic smoking includeblood leukocyte count, lipoproteins, theactivities of some enzymes, hormones, vit-amins, tumor markers and heavy metals(Fig. 4-1) (239).The mechanism underlying these changeshas not been fully elucidated. A largenumber of pyridine compounds, hydro-gen cyanide and thiocyanate are found in tobacco smoke. They can

Fig. 4-1Deviation (%) of bloodanalyte concentrations

between current smokers and non smokers, chronic

effects (239)

▼▼

400200

84CO-haemoglobin concentration %

cotinine concentration µg/L

thiocyanate concentration µmol/L

CO-Hb

cotinine

thiocyanate

200100

nonlowmoderateheavy

13

Stimulants and addictive drugs as biological influence factors

AlcoholAlcohol consumption, depending on itsduration and extent, may affect a numberof analytes. These alterations are usedin part for diagnosis and therapeuticmonitoring. Among alcohol-relatedchanges, acute and chronic effectsshould be considered separately.The acute effects (within 2– 4 hours) ofethanol consumption are decreasedserum glucose and increased plasmalactate due to the inhibition of hepaticgluconeogenesis. Ethanol is metabo-lized to acetaldehyde and then to ac-etate. This increases the formation ofhepatic uric acid formation (67).Together with lactate, acetate decreas-es serum bicarbonate, resulting inmetabolic acidosis. Elevated lactate re-duces urinary uric acid excretion. Con-sequently, after acute alcohol ingestion,the serum concentration of uric acidincreases (204).The long-term effects of ethanol ingestioninclude an increase in the serum activityof liver enzymes. The increase of g-glutamyltransferase activity is causedby enzyme induction. Glutamate dehy-drogenase as well as aminotransferas-es (AST, ALT) activities increase due to adirect liver toxic effect (57). The increasein desialylated forms of proteins inblood (i.e. carbohydrate deficient trans-ferrins) is due to an inhibition of enzy-matic glycosylation during post-transla-tional processing of these proteins inthe liver. In chronic alcoholism, serumtriglycerides increase due to decreased

plasma triglyceride breakdown. Theincreased MCV may be related to adirect toxic effect on the erythropoeticcells or a deficiency of folate (173).The data in Fig. 4-3 do not take intoaccount either the dose or the time-dependency, which underly both the acuteand the chronic effects. Enhanced diuresisis also a result of the decreased release ofvasopressin followed by increased secre-tion of renin and aldosterone (17, 115).

Addictive drugs

Addictive drugs such as amphetamine,morphine, heroin, cannabis and cocainecan influence the results of laboratorytests. Morphine causes spasms of thesphincter of oddi, thus elevating levelsof enzymes such as amylase and lipase.The biological effects of addictivedrugs on selected laboratory tests arelisted in Tab. 4- 1

Fig. 4-3Acute and toxic effectsof alcohol ingestion onclinical chemicalanalytes (115, 168,239)

osteocalcinprolactinADHcortisolANPcholesteroltriglyceridesaldosteroneLDL-cholesterolVMAMCVcholesteroltriglyceridescortisolalanine aminotransferaseestradiolepinephrinenorepinephrineaspartate aminotransferaseγ-glutamyltransferase

acute effects

chronic effects

+100-50 +200% changes

+260+1000%

Tab. 4- 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.

1

When to test?

14

Changes brought about in specimensdue to the time factor should be takeninto account in the preanalytical phase.Three questions are essential in thiscontext:

● When should a sample be taken?– Time of day– Time after last sample– Time after last meal– Time after drug etc.

● When do I require the result of thespecimen taken now?

● Can results be compared with theresults obtained at a different time indaily, monthly and yearly rhythms, ei-ther from the same patient or from areference population?

For the sake of clarity, we can differen-tiate between linear time, going fromthe past to the future, and cyclic time;both of these can influence the resultsof laboratory tests (Fig. 5-1).

Influence of circadian rhythm (217)

Several analytes tend to fluctuate interms of their plasma concentrationover the course of a day (Tab. 5- ).

Thus, the concentration of potassium islower in the afternoon than in the mor-ning, whereas that of cortisol increases

1

during the day and decreases at night(Fig. 5-2).

The cortisol rhythm may well be respon-sible for the poor results obtained fromoral glucose tolerance testing in theafternoon.

For this reason, reference intervals areactually obtained between 7 and 9a.m. The circadian rhythm can also beinfluenced by individual rhythms con-cerning meals, exercise and sleep. Theseinfluences should not be confused withreal circadian changes. In some cases,seasonal influences also have to beconsidered. Thus, triiodothyronine (T3) is20% lower in summer than in winter(82) whereas 25-OH-cholecalciferol ex-hibits higher serum concentrations insummer (162).

Analytes may change during themenstrual cycle (239)Analytes can also exhibit statisticallysignificant changes due to the biologicalchanges that occur in the hormonepattern during menstruation. Thus, thealdosterone concentration in plasma istwice as high before ovulation than inthe follicular phase. Likewise, renin can

Fig. 5-2Daily variation of

plasma concentrationsof cortisol (shaded

areas = sleep period)

Chronobiological influence

CyclicLinear(e.g. age)

Daily(circadian)

Biological (e.g.menstrual cycle)Seasonal

Fig. 5-1Linear and cyclicchronobiological

influence

300

250

200

150

100

50

00 6 12 18 h24

cortisolµg/dL


15

Timing of sampling

show a pre-ovulatory increase. Evencholesterol exhibits a significant decre-ase during ovulation. In contrast, phos-phate and iron decrease during men-struation.

Why has blood to be taken 12hours after the last meal? (49, 239)As mentioned under diet (see p. 8 –9),several metabolic products of food canincrease in venous blood or becomealtered due to post-absorptive hormonaleffects. Other analytes may be disturbedby turbidity due to chylomicronemiapresent in postprandial blood samples.

In order to eliminate these variables, a12 hour period of starvation is recom-mended before blood is sampled for theanalysis of these analytes (Tab. 5- ).

Timing with regard to diagnosticand therapeutic processesAs described in the next chapter, anumber of diagnostic procedures mayinfluence laboratory results. In order toprevent this, the timing of sampling hasto be organized to take place before

1

interfering diagnostic procedures. Like-wise, interfering drugs should be admin-istered after taking a blood sample. Onthe other hand, in drug monitoring (seep. 68) the exact timing of sampling isessential for correct interpretation ofthe drug level.

Important rules for the timing of sam-pling:

● If possible, samples should be takenbetween 7 and 9 a.m.

● Sampling should be carried out 12hours after the last meal.

● Samples should be taken beforeinterfering diagnostic and therapeuticprocedures are performed.

● In drug monitoring, consider the peakafter drug administration and the steadystate phase before the next dose.

● Always document the exact time ofsampling in the charts and requests.

But:

● A sample taken at the wrong timecan be worse than taking no sample.

● A sample whose analytical resultsarrive too late is a wasted sample.

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 50–120Cortisol (S,U) 5–8 21–3 180–200 Haemoglobin 6–18 22–24 8–15Testosterone 2–4 20–24 30–50 Eosinophils 4–6 18–20 30–40TSH 20–2 7–13 5–15 Iron (S) 14–18 2–4 50–70T4 8–12 23–3 10–20 Potassium (S) 14–16 23–1 5–10Somatotropin 21–23* 1–21 300–400 Phosphate (S) 2–4 8–12 30–40Prolactin 5–7 10–12 80–100 Sodium (U) 4–6 12–16 60–80Aldosterone 2–4 12–14 60–80 Phosphate (U) 18–24 4–8 60–80Renin 0–6 10–12 120–140 Volume (U) 2–6 12–16 60–80Epinephrine (S) 9–12 2–5 30–50 Body temp. 18–20 5–7 0.8–1.0 °C

* Start of sleeping phase

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

Sampling during infusion therapy?

16

Tab. 6- Infusions/transfusions as interfering factors and/or contaminants of laboratory diagnostictests

Infusion/Transfusion Analyte affected Trend Comments, MechanismDextran 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 positivevirus-mediated and bacterial infections

Electrolytes Potassium, sodium, magnesium ↑ Contamination

Glucose Glucose ↑ ContaminationGlucose Inorg. phosphate, potassium, ↓ Insulin

Amylase, bilirubin ↓ Up to 15 %, particularly in neonatesFructose Uric acid ↑ Metabolic effect

Citrate (blood transfusion!) pH value in blood ↓

Coagulation tests ↓↑ Inhibition

1

Implausible laboratory results afterdiagnostic and therapeutic inter-vention?

The following diagnostic and therapeuticmeasures can result in both in-vivo(frequent) and in-vitro (less common)effects on laboratory tests (79, 99, 232):

● Operations● Infusions and transfusions● Punctures, injections, biopsies,

palpations, whole-body massage● Endoscopy● Dialysis● Physical stress (e.g. ergometry,

exercise, ECG)● Function tests (e.g. oral glucose

tolerance test)● Immunoscintigraphy● Contrast media, drugs● Mental stress● Ionizing radiation

Operations

Changes in serum enzyme activitiesare frequently so great that specifictargeting of an organ is no longerpossible. The elevation in acute phaseproteins (e.g. C-reactive protein (CRP),fibrinogen) at the beginning of thepostoperative phase is accompaniedby a decrease in albumin; this cannotbe explained alone by haemodilution.

Transient elevations in urea concentra-tion in serum/plasma (up to 60 mg/dLor 10 mmol/L) as well as a decrease incholesterol are very frequent in the firstpostoperative days whilst the creatinineconcentration remains normal. This maybe due to protein breakdown subsequentto gastro-intestinal tract surgery as wellas to bleeding in the lumen of thebowel, e.g. in the case of a stress ulcer.

17

Sequence of diagnostic and therapeutic procedures

Infusions, transfusions

Haemolysis and hence the concentra-tions of free haemoglobin and potassi-um, as well as the activity of lactate de-hydrogenase in plasma obtained fromconserved blood, increase with the ageof the transfused conserved material.

Contamination of laboratory samplesby infusion solutions is the commonestand often the most relevant form of preanalytical interference in the hospital (228, 242) (Tab. 6- ).

Blood should never be collected proxi-mal to the infusion site.

Specimens should be collected from theopposite arm. A certain period of timeshould be allowed to elapse followinginfusion therapy (Tab. 6- )

Tab. 6- Recommendations for schedulinginfusions and blood sampling

Infusion Earliest time of bloodsampling in hours aftercessation of infusion

Fat emulsion 8Carbohydrate-rich solutions 1Amino acids and protein hydrolysates 1Electrolytes 1

It is recommended that the laboratorybe informed of when and what type ofinfusions were carried out and whenblood samples were taken.

Sampling from catheters

If samples are to be taken from intra-venous and intraarterial infusioncatheters, the cannula should be rinsedwith isotonic saline commensurate withthe volume of the catheter. The first 5mL of blood should be discarded be-fore a blood sample is taken.

2

2

1

Sampling for coagulation tests fromheparin-contaminated catheters is par-ticularly critical. For heparin-dependentmethods (thrombin time, APTT), it isrecommended that an amount of bloodequivalent to twice the volume of thecatheter be discarded; the blood firsttaken after this should be used for non-haemostaseological investigations andthe subsequently obtained citrated bloodonly used for determining heparin-insensitive analytes: Prothrombin time,reptilase time, fibrinogen according toClauss, AT III, fibrin monomers. It isimportant that before transferring bloodto the sampling vessel containing sodiumcitrate solution there is no lengthy pauseduring which the blood in the catheteris allowed to “stand“.

Mental stress

The importance of mental stress onlaboratory results is frequently under-estimated (anxiety prior to blood sam-pling, preoperative stress, etc.). Incre-ased secretion of hormones (aldoste-rone, angiotensin, catecholamines, cor-tisol, prolactin, renin, somatotropin, TSH,vasopressin) and increased concentra-tions of albumin, fibrinogen, glucose,insulin, lactate and cholesterol havebeen observed.

Fig. 6-1

Sampling in the supine or upright position?

18

Posture

It is a well-known fact that body postureinfluences blood constituent concentra-tions. This is caused by different me-chanisms. First, the effective filtrationpressure (e.g. the difference betweencapillary pressure and colloidal osmoticpressure in plasma) increases in thelower extremities when changing fromthe supine to the upright position. As aconsequence, water is moved from theintravasal compartment to the interstitium;this reduces the plasma volume byabout 12 percent in normal individuals.Blood particles with a diameter of morethan 4 nm are restrained by membranesand cannot follow this volume shift. Achange from the upright to the supineposition leads to a decrease in the effec-tive filtration pressure and hence to a vol-ume shift in the reverse direction (176).A change in plasma volume leads toan apparent concentration change incells, macromolecules and protein-bound small molecules. Most low mo-

lecular weight compounds show nochange in their apparent concentrationswhen changing from the upright to thesupine position. As osmolality is mainlymediated by such compounds, the firstis only modestly affected by changes inplasma volume (1–2%). Because ofpartial protein binding, the concentra-tions of free and bound calcium areaffected in a different manner. Whilstthe concentration of free calcium isindependent of posture, total calciumincreases by 5–10 percent whenchanging from the supine to the uprightposition (172). Other changes are dueto altered blood pressure which in turncauses secretion of vasoactive com-pounds. In addition, the metabolic con-sequences of regulatory changes due topostural changes may alter body fluidcomposition.The effects of posture on analytes invenous anticubital blood are shown inFig. 7-1. As expected from the describedmechanism, most cellular and macro-molecular analytes decrease between 5and 15% compared to the supine po-sition. These effects can be more pro-nounced in patients with a tendency toedema (cardiovascular insufficiency, livercirrhosis). Reduction in plasma volumeinduces a decrease in blood pressurewhich in turn leads to increasedsecretion of renin, aldosterone, norepin-ephrine and epinephrine. A fall in bloodpressure causes decreased secretion ofatrial natriuretic peptide which leads todecreased plasma concentrations (211).An example of the metabolic changesbrought about due to posture is theurinary excretion of calcium which in-creases during long-term bed rest (seeFig. 12-2, p. 28).

Tourniquet

What happens when a tourniquet iskept on during sampling? A tourniquet

% increase

haemoglobinleucocyteshaematocriterythrocytes

total calciumaspartate aminotransferasealkaline phosphataseimmunoglobulin Mthyroxineimmunoglobulin Gimmunoglobulin Aalbumintotal proteinapoprotein BcholesterolLDL-cholesteroltriglyceridesHDL-cholesterolapoprotein AI

aldosteroneepinephrinereninnorephinephrine

% increase10 20

50

Fig. 7-1Increase (%) of plasma

concentration ofvarious analytes when changing from supineto an upright position(55, 131, 232, 239)

19

Effects of posture and tourniquet

is usually applied to facilitate findingthe appropriate vein for venipuncture(see p. 20). Using a pressure below thesystolic pressure maintains the effectivefiltration pressure inside the capillaries.As a consequence, fluid and low mo-lecular compounds are moved from theintravasal space to the interstitium.Macromolecules, compounds bound toprotein and blood cells, do not pene-trate the capillary wall so that their con-centration apparently increases whilethe concentration of low molecular sub-stances is unchanged.Fig. 7-2 shows the changes of differentanalyte concentrations (93). The altera-tions of low molecular weight analytesobserved following 6 min. of constric-tion are in the range of ±3 percent andtherefore within the range of analyticalimprecision. However, it has been shownthat constriction of the forearm musclescauses an increase in serum potassiumconcentration. Therefore, during veni-puncture for potassium determination,repeatedly clenching and unclenchinga fist should be avoided and a superfi-cial vein selected (45, 197). A venosta-sis of two minutes did not change theblood lactate concentration (mean+2.2 %), but decreased the pyruvateblood concentration significantly (mean–18 %) (108). The extent of changes inhigh molecular weight analytes de-pends on the duration of constriction.Fig. 7-3, for example, demonstrateschanges of lactate dehydrogenase ac-tivity and total protein concentration.The greatest effects are observed within5 minutes, little change taking placethereafter (58). Constriction times of oneminute with subsequent release of thetourniquet have no consequences onplasma serum analyte concentrationsand coagulation factors.To reduce the intra- and interindividualvariance of laboratory results a stan-dardised sampling procedure is an im-

portant prerequisite. The following con-ditions should, whenever it is possible,be fulfilled: a preceding phase of restand fasting, the same body position,daytime and tourniquet applicationtime, avoidance of repeatedly clench-ing and unclenching a fist. The tourni-quet application time should not belonger than one minute. During a run-ning infusion the phlebotomy should beperformed on the opposite arm, in anycase not proximal from the running in-fusion. If it is necessary to repeat sam-pling for any other reason, the phle-botomy should also be performed onthe other arm (176, 177).

Fig. 7-2Change (%) in serumconcentration ofvarious analytes aftera tourniquet applica-tion time of 6 min (93)

alanine aminotransferasecreatine kinasebilirubinlactate dehydrogenasealbuminalkaline phosphatasetotal proteincholesteroltriglyceridesaspartate aminotransferasecalciumerythrocyteshaemoglobinhaematocrituric acidsodiumpotassiumchloridecarbon dioxidecreatinineurealeukocytesinorganic phosphateglucose

2 4 6 8 10 12 %–4 0–2

In comparing laboratoryresults, reference inter-vals should be obtainedunder identical condi-tions with regard tobody posture (176).

90

85

80

75

70

120

110

100

0 2 5 10 15 min

total protein(g/L)

lactate dehydrogenase(U/L)

Fig. 7-3Change in total proteinconcentration ( ) andlactate dehydrogenaseactivity ( ) in serumduring a 15 mintourniquet applicationtime (58)

What site for sampling blood?

20

The site of collection such as vein,capillary or artery has a bearing onthe procedural aspects of specimen col-lection (205).Specific NCCLS documents address pro-cedures for the collection of blood spe-cimens by venipuncture (156), skin punc-ture (157) and arterial puncture (158).

Phlebotomy

Critical steps in phlebotomy involve notonly preparation of the patient forblood collection and proper collectionof the specimen, but also other stepsamongst which are proper identificationof the patient. This avoids patient mix-upand ensures patient safety. Fig. 8-1visualizes the steps to be taken in blood collection using evacuated tubes.● Identification: Match the patient's

test order form with the patient number,bar code or wrist band number.

● Position: Position of the patient(sitting or recumbent) for venipuncture.

● Materials: Ensure blood collectionequipment (needle, collection holder,tubes) is at hand.

● Inspection: Inspect patient’s arm, select a vein while the patient’s fist isclenched.

● Disinfection: Clean the venipuncture site.● Vein Exposure: Apply a tourniquet.● Puncture Collection: See Fig. 8-1.● Mixing: Mix the blood in tubes

containing additives or clot activators.● Prevention of Bleeding: Apply gauze to

the venipuncture site while removingthe needle. Apply a bandage to the pa-tient’s venipuncture site.

● Disposal: Dispose of the needle in asafety disposal unit.

The order of collection of tubes is im-portant when multiple tubes of bloodare collected (Tab. 8- ).

Quality of sample: Examine to see whether the tubes areoverfilled or underfilled.

Overfilling tubes intended for haematol-ogy determinations can cause spuriousresults due to the absence of a bubbleat the very top which will prevent prop-er mixing of the specimen on rockertype mixing devices (166).

For certain procedures, the anticoagu-lant to blood ratio is critical (see p. 52).

If multiple blood collection tubes are tobe collected (156), the following orderof collection is recommended:

1

Fig. 8-1Steps in blood

collection

15o

Reverse the positions ofthe hands as soon as theneedle is in place. Pressthe tube home with thethumb of the right hand,the index and centerfingers being supportedon the flange of theholder.

The blood is drawn upby the vacuum and flowsinto the tube (*) on itsown. Release the tourni-quet immediately withyour left hand still hold-ing the holder.

Withdraw the tube withthe right hand, support-ing the thumb on one ofthe flanges of the holder.

Agitate the tube verygently, turning it overseveral times to ensureproper mixing with theanticoagulant.

If multiple blood samplesare to be taken, insert asecond tube and repeatthe operations startingfrom b.

Penetrate the skin, hold-ing the complete unitbetween the thumb andindex finger of the righthand.

a b c

e fd

* 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 with-drawing 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

21

Phlebotomy, arterial puncture and sampling from catheters

Tab. 8- Recommended sequence of collectingvarious blood specimens

1. Blood culture Blood2. Non-additive tube Serum3. Citrate Plasma4. Heparin Blood5. EDTA Blood/plasma6. Glycolytic inhibitor Glucose/lactate

If a blue stoppered citrate tube intendedfor coagulation testing is to be drawnfirst, a 5 mL discard tube should be filledto eliminate possible thromboplastin con-tamination from the venipuncture site.

How much blood is needed?The volume of blood collected from apatient should be kept to a minimum toavoid blood loss that could render thepatient anemic. The term investigationalanemia has been coined to refer toblood loss due to repeated venipunctures.Often, too much blood is drawn as isapparent from a Mayo Clinic studywhere it was reported that for routinecollections, an average of 45 times therequired volume of specimen (range 2to 102 times!) was withdrawn, while formicrocollection an average of 7 timesthe required volume of specimen (range1 to 20 times) was withdrawn (40). Inan earlier study it was reported that47% of patients who required bloodtransfusions had a phlebotomy-relatedred blood cell loss of more than 180 mL,an amount equivalent to 1 unit ofpacked red blood cells (198).It was recommended (71, Annex) tocollect twice the amount of blood need-ed for analysis. The amount of bloodneeded may be calculated from the an-alytical portion needed, the dead spaceof the analytical system and the sampletube using the following formula:Blood volume needed = 2 x [number ofrepetitive tests x (analytical volume +dead space of the analytical system) +

1 dead space of the secondary tube] +dead space of primary tube. This for-mula assumes that 50 % of blood vol-ume can be used as analytical volume(71, Annex).

Sampling from arteryParticular care should be taken whenblood is collected from an artery (158).The common sites of arterial punctureare the femoral artery, brachial arteryand radial artery. Other sites include scalparteries in infants and the umbilical arter-ies during the first 24 to 48 hours of life.Arterial puncture is necessary whenvenous blood does not permit the mea-surement of the relevant concentrationof the desired analyte (e.g. blood gases,pH). Arterial puncture can be performedeither singly by inserting a short-beveled, sharp needle into an artery. Asyringe can be attached to the needleeither directly or by way of tubing withan adapter such as in a winged infusionset. Arterial blood can be collectedwithout suction when using 23-gaugeor larger needles, allowing the pressurein the artery to force the blood into thesyringe.

Sampling with catheterContinuous or repeated sampling ofarterial blood can be performed byleaving a permanent needle, cannulaor catheter in the artery or central vein.Care should be taken to ensure that noclot is formed at the tip or in the lumenof the catheter. Between samples, ananticoagulant (preferably heparin)should be used to flush the needle.When sampling with a venous catheter,the coagulation tube containing sodiumcitrate should be filled before a he-parin-containing tube is collected. Thefirst few mL of blood, representing 1 to2 volumes of the catheter, should bediscarded to avoid contamination withanticoagulant.

Blood from the skin

22

Skin puncture is the procedure of choiceif a small amount of blood is to be takenfrom a pediatric subject. In adults,capillary blood is used for blood gases,glucose and lactate. There are differ-encesbetween capillary and venous blood,especially in oral glucose tolerancetesting.

Blood obtained by skin puncture iscomposed of a mixture of blood fromthe arterioles, venules and capillaries;it may also be diluted with interstitialand intracellular fluid.

The relative composition of skin punctureblood will depend on variables such asblood flow to the skin during collection.Warming the puncture site prior toblood collection in effect arterializesthe blood in the skin (157).

Sites for blood collection are illustratedin Fig.9-3. They include the palmar sur-face of the distal phalanx of the fingerand the lateral or medial plantar surfaceof the heel (157).

Finger puncture should not be performedon infants since there is a likelihood ofinjuring the bone. There is a linear rela-tionship between the volume of bloodcollected and puncture site penetrationdepth (7). Therefore, the lancet shouldbe selected according to the site punc-tured and the amount of blood needed(Fig. 9-1).

The depth of incision made to an infant’sheel is critical since puncturing deeperthan 2.4 mm on the plantar surface ofthe heel of small infants especially candamage the calcaneus or heel bone.This can be avoided by using semi-automatic disposable lancets (Fig. 9-2).After selection of the skin-puncture site,the site should be cleaned with a 70%aqueous solution of isopropanol priorto puncture (125). After drying the sitewith a sterile gauze to ensure thatresidual alcohol has been removed(since it would otherwise cause haemol-ysis), skin puncture should be per-formed with a disposable lancet. Theuse of other disinfectants to clean thepuncture site should be avoided as thiswould spuriously elevate the results foruric acid, phosphorus or potassium (225).

The first drop of blood that flows afterskin puncture should be discarded bywiping with gauze, since this drop is

Fig. 9-2Genie™ lancet convenient,

push-buttonoperation

automaticbladeretraction

safe,uniformpenetrationdepth

single use2

1 3

4

Fig. 9-1Penetration depth of

Genie™ lancet

▼

1.25 mm 2.25 mm 2.0 mm 1.5 mm1.0 mm

23

Capillary sampling

likely to be contaminated with tissue flu-ids. By application of gentle pressurebut without milking or massaging thearea around the puncture site, freeflowing drops of blood should be col-lected by touching the drops with thetip of a collector top and letting themflow by capillary action into a properlylabeled microcollection device.

Fig. 9-3 illustrates a typical microcollec-tion technique. In the event that dropsdo not flow freely from the collector topinto the microcollection tube, the tubemay be gently tapped to facilitate theflow of drops of blood into the tube.Upon completion of blood collection, thetube should be firmly secured. Additive-containing tubes should be well mixedafter specimen collection by gently in-verting the tube approximately 10 times.

Collection of blood specimens intoheparinized capillary tubes intended

for blood gas analysis should be madeafter warming the puncture site with atowel soaked in running water at atemperature not greater than 42 °C tobring about arterialization of the punc-ture site. The capillary tubes should befree of air bubbles after collection.On conclusion of specimen collection,the puncture site should be pressedwith a sterile gauze or swab and heldin place until bleeding stops. As asafety measure, it is advisable not toapply adhesive bandages over thepuncture site of infants and children,not only out of concern that the adhe-sive may cause irritation but also dueto the fact that the bandage could be-come loose and be swallowed by thechild.

The safety lancets used for skin punc-ture should be deposited in an appro-priate puncture-resistant safety con-tainer intended for such a purpose.

Fig. 9-3Capillary blood collec-tion with a typical mi-cro collection device

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.Depress the plunger and release. Remove lancet and dispose into a con-tainer for sharp objects.

d) Wipe away the first drop of blood. Position tube at puncture site and chan-nel blood flow down the center of the collector into the tube. Do notsqueeze the area.

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.

YES

YESNO

YES

MAXIMINI

EDTA

500250 500

250

250500500

250

Did the lab mix up my sample?

24

As a minimal require-ment the sample entering the lab shouldcontain the following information:

• Name, prename

• Date of birth

• Identity number of sample, or patient orrequest

• Ward number (sender,name of ordering physician)

• Time of sampling (day,hour, min)

Everyone concerned with diagnosticprocedures is aware of the problem ofsample identification. The mixing up ofpatients’ names, requests, samples or testresults can have serious effects on patienttreatment and should therefore be con-sidered as maltreatment. This chapterbriefly summarizes the present state of theart of patient and sample identificationduring the preanalytical phase (18).

Name or number?

When admitted to a hospital, a patienthas to be identified by many peopleincluding nurses, doctors, techniciansand other persons. The same is true forany sample taken from the patient andtransferred to an in-house or externallaboratory. As can be seen in Fig.10-1,communication between all parts of thediagnostic process requires transfer ofthis patient identification. A name usedto identify an individual has proved tobe insufficient in transferring the identity.Name, prename and date of birth isthe most frequently used combinationof information for identification. In orderto reduce the amount of informationinvolved, a patient number is allocatedin many hospitals. This number cannot beused for any other individual. Ideally,

this number is printed together with thename on any request, sample andreport. Ward, room and bed numbersmay be of additional help. This is espe-cially useful if sampling is not done bythe same person as the one orderinglaboratory tests.

Since such a large amount of informationcannot be entered on small labels, aseparate request form usually containsall the basic information together withthe test orders linked to the sample bya number or code only.

Fig. on the back of the above text illus-trates an example of a request form con-taining a sufficient number of labels forall possible samples, used on sampletubes (Fig.10-2). Alternatively, a packageof preprinted, self-adhesive labels withthe patient identification may be provid-ed on admission to hospital (Fig.10-3).

Techniques of identification

Upon arrival in the laboratory, the patienthas to be identified from the informationprovided. This can be done visually byreading the name and ward from thelabel and request form, transferring thisinformation to the laboratory chart andall subsamples. The latter requires thegeneration of a sub-numbering systemwhich is linked to the individual and the day of analysis (usually no morethan three digits long).

Many institutions, however, use electron-ic means to transfer identification data:this can be done on-line using a hospitalinformation system which transfers thebasic data of the patient together withthe request directly to the laboratorycomputer. In this case, the samples ar-riving in the laboratory have to belinked to a given request by bar-code or alternative code system fixed to the

Fig. 10-1Information flow

during a laboratorytest in a hospital.

HIS = hospitalinformation system

LIS = laboratoryinformation system

pre-identifiedadhesive-backedlabels

phlebotomy at ward

patient sample

identified sample

LIS

HISWard

Lab

doctor orders lab tests

printing accessiom number onadhesive-backed labels

patient sample identifiedand provided withaccession number

technique with manualreading of identification

results

results enteredby a VDT

work listedition

25

Techniques of sample identification

Fig. 10-2Request number onadhesive labels forsample tubes

Fig. 10-3Patient’s label provi-sion at hospital admis-sion and their use inthe laboratory

samples. In the example given in Fig.10-2, the number on the sample is iden-tical to the number of the request-formwhich is then electronically linked tothe patient by the laboratory computer(Fig. 10-3).

Recently, more sophisticated systemshave been suggested and will be widelyused in future: a two-dimensional barcode (Fig. 10-4), or transponder-chipsfixed to the sample can contain all thenecessary information including re-quests and patient information.

Direct versus indirect sampleidentification in the laboratory

The identity of the patient is unequivo-cally linked to the sample container atthe collection point of the sample, pro-viding the primary sample is maintainedthroughout the analytical process. Thisneeds serum/plasma separators to keepthe analytical sample (plasma/serum)in the primary sample (blood) tube sothat it can be identified directly by thereader of the analytical instrument.

If such a reader is not available or sub-samples have to be distributed, a samplingdevice may be used to automaticallydefine the positions of each sample in

each analyzer used. This kind of identi-fication is called indirect identification(by location) (128). Whereas direct iden-tification mechanisms allow the sampleto be identified at any position and anytime, indirect identification by positioncan be done only with the aid of a po-sition list or a computer system. In addi-tion, any sub-sampling has the inherentdanger of additional sample mix-up.

● Any sample should be identified un-equivocally before being analyzed.

● Direct identification procedures fromprimary samples should be used inpreference to indirect identificationand subsampling, to reduce misiden-tifications.

● Any sample whose identity and ori-gin is not sufficiently documentedshould be processed to a stable form(serum, plasma, closed in refrigera-tor) and missing information addedbefore analysis is performed.

ward

surgery 06 85John Smith 0001Gluc UR CH

laboratory reception

hospital admission

laboratory workstation

accessionnumber

▼

Fig. 10-4 Two dimensional bar-code in compari-son to unidimensional bar-code.

▼ ▼

A precious sample

26

How to collect CSF?

Puncture sitesCSF and serum form an inseparable unitin modern diagnostics; for this reasonCSF and serum should be collected asclosely together as possible (103). Thepuncture site is generally lumbar, but also

ventricular, suboccipital or via a shuntand should be noted.

The puncture site should be marked,and the area disinfected. Treatment ofweals with a local anesthetic is desir-able for the patient. Puncture should besaggital and sloping upwards (20 °)(Fig. 11-1).

Amount requiredThe quantity taken depends on theclinical situation and is not so critical inadults; CSF is very rapidly replenished

(50 mL/day in adults). Particularly whensearching for tumor cells, it is importantto obtain as much CSF as possible (upto 30 mL being optimum in adults) (Tab.11- ). The sampling time should be aslong as possible, using a needle as fineas possible, to avoid headache follow-ing puncture.

Which specific aspects of CSFsampling are important?

The fasting patient should be seated orasked to lie on his/her side on a flatsupport. The patient‘s back should bebent forwards and the position securedby an assistant. The musculature shouldbe as relaxed as possible (Fig. 11-1).The time at which the sample is takenshould be noted, together with infor-mation on any initial treatment (e.g. inbacterial meningitis).

It is recommended that an “atraumatic“pencil-shaped needle (0.7 mm outer di-ameter), as designed by Sprotte andWhitacre (Fig.11-2), be used instead ofa 22G needle with a Quincke cut so asto avoid post-puncture syndrome(headache) (24).

In order to avoid contamination CSFshould be obtained and transported inclosed tubes.

The CSF should be placed, under asepticconditions, in separate colorless poly-styrene tubes with stoppers (sterile formicrobiology and dust-free for cytologyor clinical chemistry). For cytology, theuse of additives such as EDTA and fluo-ride should be avoided. After a samplehas been obtained, the needle shouldbe removed and the wound coveredwith a plaster. Patients should be keptlying on their stomachs for at least 30min to avoid subsequent leakage.

1Fig.11-1Sampling of

cerebrospinalfluid

Fig.11-2“Atraumatic” pencil –

shaped needle

27

Cerebrospinal fluid (CSF)

Tab. 11- Sequence and quantity of CSFrecommended

Fraction Adults InfantsDiscard the first 0.5 mL and allartificially sanguineous CSF

Microbiology* ~ 2 mL ~ 1 mLCytology > 10 mL > 1 mL

Supernatant used for (tumor cells!) (tumor cells!)clinical chemistryTotal 12 mL 2 mL

*Before commencing chemotherapy, or 2–3 days after its withdrawal.

What about storage and transport?Most important of all: As soon as possible after the samplehas been taken, it should be sent to thelaboratory by courier.

CSF is not particularly “cell friendly“.Transport in an icebath over 200 km bycar (approx. 3 hours) is possible. Sta-bility data of individual componentsare given in the Annex. Further recom-mendations are given in Tab. 11- .

Tab. 11- Recommendations for transport andstorage of CSF

Up to 1 hour Do not coolUp to 3 hours Transport on ice

Never deep freezeNo additivesNo partial fixation

Long-term storage After centrifuging off cells,immediately cool to –70 °C inglass or polypropylene vesselsthat can be tightly closed.

For cytological investigations, insteadof original CSF, send cytocentrifugationpreparations to the laboratory. Thisshould be prepared close to the patientusing a special centrifuge (centrifugationfor 20 min at 180 g). These samples

2

2

1 are stable for 4 to 6 days at room tem-perature or – when fixed in acetone –for 3 to 12 months at –70°C (immuno-cytology depending on the antigen)(237).

Special hints for CSF sampling

● Sampling in several portions meansthat the concentration gradients thatalways exist (e.g. for albumin and IgG)are not taken into account. This can beavoided if the entire volume is first col-lected in one tube, well mixed and thendivided into aliquots.

● Gloves dusted with talcum powdershould not be used when withdrawingCSF, as CSF cytology is thereby invali-dated.

● The presence of up to 6000 leuko-cytes/µL should not alter the lactateconcentration within 3 hours at roomtemperature (120).

● Deep-freezing of CSF is better carriedout at –70°C than at –20°C since, forexample, oligoclonal protein bandsslowly disappear after storage over6 months at –20 °C.

A sample that is nearly always available

28

UrineA review of the ten most popular errorsin the analysis of urine revealed thatsome of the problems were of a prean-alytical nature: samples were too old,sampling vials had been contaminated,the samples were not homogeneousand preanalytical factors had not beenadequately taken into account when in-terpreting test results (59, 109). The qual-ity of the collection vial is a critical fac-tor in this context. The ideal containerfor any urine specimen is a wide-mou-thed bottle of appropriate size (Fig.12-1). Urine containers used should eitherbe disposable or, if not, detergentshave to be reliably removed prior touse. If the urine is to be analyzed bacte-riologically, containers have to be ster-ile. In protein and hormone analysis,analyte absorption to the vessel wall

should be avoided. The different kindsof sampling are given in Tab. 12- . For pediatric and newborn patients,urine specimen collection bags withhypoallergenic skin adhesive should beused. First, the pubic and perineal ar-eas should be cleaned with soap andwater. Then, the adhesive should bepressed all around the vagina or thebag fixed over the penis and the flapspressed to perineum. The containershould be checked every 10–15 min(153). Urine samples obtained in themorning offer several advantages. Ahigh degree of osmolality indicates anintact concentrating ability on the partof the kidney. Urine samples obtainedin the morning are particularly useful inidentifying mycobacteria. Deviationsdue to diet, physical activity and pos-ture are diminished. Fig.12-2 shows the

1

Fig. 12-1Containers for urine

specimens for qualita-tive and quantitative

testing

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

1. Random or spot urine Qualitative chemical determinations2. First morning urine Cellular constituents and casts3. Second morning urine Quantitative determinations related

(7 – 10 a.m.) to creatinine4. 24 h urine Quantitative determinations

1

%

240

200

160

120

100

-4 -2 0 2 4 6 8 10

immobilization

perc

ent o

f exc

retio

n

weeks

Fig. 12-2Urinary excretion of calcium during a six-weekimmobilization period

▲

29

Urine and saliva as diagnostic probes

increase of the urinary excretion of cal-cium to 240% during a six week immo-bilization period (240).

Fig.12-3 gives an example of the influ-ence of diet on the urinary excretion ofelectrolytes and some substrates. Underthese special conditions, five healthyvolunteers received only distilled waterduring a four-day voluntary period ofstarvation. Most analytes showed a re-duced level of excretion. Only ammoniaexcretion is increased as a consequenceof the metabolic acidosis induced bystarvation (106).

For quantitative analysis, one shoulduse timed urine, preferably in the formof 24-hour urine collection (104). Mid-stream catheterized or suprapubicpuncture urine specimens are recom-

mended for bacteriological investiga-tions (59, 109).

In Fig.12-4, the collection of a mid-stream urine (clean-catch urine) isshown schematically. Mid-stream urinecontaining bacteria >105/mL is consid-ered to indicate significant bacteruria.Analysis of urine samples should ideallyproceed within one hour followingsample generation (except in the caseof 24-hour urine samples). A short pro-cessing time is of particular importanceonce a morphological evaluation of thelabile urinary sediment constituents hasto be performed. Thus, the stability oferythrocytes and leukocytes in urine isdependent on both pH and osmolality.A pH above 7.5 and an osmolalitybelow 300 mosm/kg results in rapiddegradation of these cells (165). Other

sodium

potassium

calcium

magnesium

chloride

inorg. phosphate

sulfate

creatinine

urea

ammonia

-100

-50

212

144

% changes

50

100

Fig. 12-4Collection andsampling of mid-stream urine (clean-catch urine)

Fig. 12-3Influence of 4-daystarvation on urinaryexcretion of elec-trolytes andsubstrates. Change (%)during ( ) and oneday after thevoluntary starvation( ) related to thestarting value (106)

A sample that is nearly always available

30

No single additive is available that stabi-lizes all classes of compounds.

For quantitative determination, storagewith a stabiliser at room temperature isrecommended. If the urine samples arestored at 4–6°C for calcium, magne-sium, oxalate and phosphorus determi-nation, acidification (pH 1.5–2.5) and,for uric acid, alkalization (pH > 8.0) isnecessary. For special analytes, differ-ent types of preservatives are neces-sary (see Tab. 12- ).

Saliva

Saliva can be used for analyzingvarious compounds such as steroidsand drugs. Samples obtained can bederived from one gland only (gland-specific saliva) or be a mixture ofproducts from different glands (mixedsaliva) (Fig. 12-6). The latter specimenis used for routine procedures. Differenttechniques are employed to obtain salivafrom the oral cavity (77). Several collec-tion devices have been developed tostandardize the collection of saliva forhormone and drug monitoring. Tab. 12- provides a selected listing of thepresently available devices and adsorb-

3

2

analytes tend to form crystals at physio-logical pH (calcium, oxalate, uric acid)or are decomposed if not adequatelystabilized (glucose, urea, citrate).Fig.12-5 shows the changes (%) ofvarious analytes after a storage of two,four and six days at room temperaturewithout additives. Citrate, glucose,oxalate and magnesium are unstable.Addition of sodium azide in a finalconcentration of 10 mmol/L urine forthe same time and temperature stabi-lized all these constituents for 6 days atroom temperature (186). Tab. 12-lists a number of additives which areused to stabilize compounds in urine.

2

Fig. 12-5Influence of storage

time on the recoveryof various analytes inurine samples withoutadditives and storageat room temperature(% changes from thestarting point) (186)

oxalateglucose

creatininecitrate

albumin

2 days

4 days

6 days

t-proteininorg. phosphate

magnesiumcalcium

uric acidurea

100

50

% d

ecre

ase

sodiumpotassium

Tab. 12- : Urine preservatives (59, 91)

Preservative Analytes stabilized

Thymol, 5 mL of a 10% solution in 2-propanol Most constituents

Sodium azide, 10 mmol/L urine Glucose, urea, uric acid, citratepotassium, calcium, oxalate

Hydrochloric acid, 25 mL 6 mol/L per Catecholamines and metabolites,24-hours urine volume 5-hydroxyindolacetic acid,

calcium, magnesium, phosphate

Sodium carbonate, 2 g/L urine Porphyrins, urobilinogen

Urine pH > 8.0 Uric acid

2

31

Urine and saliva as diagnostic probes

ing materials. No one device can beconsidered ideal for all purposes. Devi-ces using materials adsorbing saliva areeither contaminated with substances in-terfering with the measuring procedureor the drug itself is adsorbed by thematerials to varying degrees depend-ing on the device used. Recoveries ofdrugs have recently been reported torange from 59 to 107% (78).

Saliva has several advantages in drugmonitoring compared to blood. Goro-discher has reported that 85% of pa-rents and 50% of children indicated apreference for saliva sampling over ve-nous blood withdrawal (64). The easeof collection of saliva makes it ideal forself-testing at home and in cases whereblood sampling is difficult (e.g. in new-borns). Limitations in sampling posedby saliva are related to viscosity and insome cases to the difficulty in obtainingsufficient volume. The saliva/plasma ratioof drugs of abuse has been reviewed(78).

Tab. 12- : 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 deviceOmni-Sal Saliva Diagnostic Systems Adsorbing pad with fluid volume indicator Separation by pressure through a

filter into a buffer containing tubeOrapette Trinity Biotech Rayon ball sampling Expulsion by screwing a pistonOra Sure = Epi Screen Epitope Pad on "lollipop” stick Centrifugal tube with

antimicrobial bufferAccu Sorb™ Avitar Technologies Polymer pad fixed on the closure Squeezing manually (milking)

into a plastic tubeOral Screen Avitar Technologies Polymer foam cube sampling Squeezing by pressure on the

surfaceAbusa-stick Chem-Elec Saliva swabAlcoscan Lifescan Saliva swabQ.E.D. (used SCT Technologies Cotton swab on a stick Squeezing by pressure on thefor saliva alcohol test) surface

3

Fig. 12-6 Collection of saliva from different locations of the oral cavity. In theupper part 4 cotton rolls connected by a thread were placed above thetongue. At the bottom 3 shortened cotton rolls connected by a thread wereplaced below the tongue. On the left and right side metalline strips areshown which are wrappped in polyethylene foil with a rectangular openingin the area of the orificium of the ductus parotidis. The 4 parts were placedinto the oral cavity and left there for 9 minutes without moving the tongue.Saliva was collected from the strips and the cotton rolls by centrifugation inSalivettes® (78)

Plasma or serum?

32

can becentrifugedimmediately

store for 30-–45 minundisturbed

and, if possible,in the dark;centrifuge

Serum is obtained from whole blood bycentrifugation after completion of theplatelet and clotting factor coagulationprocess. Serum must therefore be re-garded as an artifact. By definition, it is devoid of clotting factors but isenriched with the cellular componentsof platelets and metabolic products.

Plasma is the virtually cell-free super-natant following centrifugation of wholeblood, the coagulability of which isinhibited by the addition of anticoagu-lants during or immediately after sam-

pling. Anticoagulants inhibit clot forma-tion through various mechanisms (72,Annex). The required concentration ofanticoagulants and their composition isdescribed in an international norm(89).

For some analytes, there are diagnosti-cally relevant differences between theresults obtained from serum and thoseobtained from plasma (see Tab.13-and Fig. 13-1). A linear correlation hasbeen shown between the difference inserum-plasma potassium and phos-phate and the number of platelets inblood (Fig.13-2). Some constituentsfound in high concentrations in plateletscan therefore not be correctly deter-mined in serum, e.g. acid phosphataseactivity, neuron specific enolase,dopamine and serotonin (69).

Differences between the values ob-tained in serum and plasma are due tothe following physiological and technicalreasons:● The analyte may be consumed duringclotting: fibrinogen, platelets, glucose.● The analyte may be released fromcells during clotting: potassium, lactatedehydrogenase, phosphate, ammonia,lactate.● The anticoagulant may interfere withthe assay or contaminate some assays:

1

Fig. 13-1Plasma serum differen-

ces obtained in 4 mLseparator tubes (226).

Ratio of the median dif-ference between serum

and plasma and thecoefficient of variation

of the analytical proce-dure used

Fig. 13-2Dependence of plasma-

serum difference inpotassium

on platelet count inblood (121)

1.81.61.41.21.00.80.60.40.20.0-0.2

200

400

600

800

1000

1200

1400

1600

platelet count (x 109/L)

K diff

(mm

ol/L

)

Tab. 13- : Analytes with diagnostically relevant serum/heparinized plasma concentration differences and their main causes (226)

Analyte % change in comparison Main cause of the serum/to the mean in plasma plasma difference

Potassium +6.2 Lysis of the cells, particularlythe platelets*

Inorganic phosphate +10.7 Release from cellular elementsTotal protein –5.2 Effect of fibrinogenAmmonia +38 Thrombocytolysis, hydrolysis of

glutamineLactate +22 Release from cellular elements

* Also causally responsible for high potassium values in serum are – in addition to thrombocytolysis – haemolysisand extreme leukocytolysis (when leukocyte counts are > 50 G/L). Concerning the platelet influence in wholeblood, the following applies: an increase in the platelet count by 100 G/L means an average increase of 0.11mmol/L in the difference between the serum and plasma potassium values.

1

total proteinalbumin

TSHtransferrin

ASATcreatine kinase

total bilirubinsodium

ironcholineesterase

alkal. phosphatasedirect bilirubin

triglyceridesLDH

inorg. phosphatepotassium

γ−glutamyltransferasetriiodothyronine

lactate

high

er in

pla

sma

0-5 5 10

high

er in

ser

um

ratio of serum – plasmaplasma x cv x 100

▲

Plasma Serum

Anticoagu-lants

No anticoag-ulants

Blood

33

Differences to be considered

γ-glutamyl transferase, lithium in flamephotometry, when calibrated with lithium.

● Methodology of the respective deter-mination, including for example the typeof measuring instrument used (mono-chromatic/bichromatic measurement)(80) may cause interference. This in-cludes interference of fibrinogen withsome heterogeneous immunoassays.

What advantages does plasmahave over serum?The recommendations published by theWorking Group on Preanalytical Quali-ty (72, Annex) has described the ad-vantages and disadvantages of usingplasma versus serum:

1. Time saving. Waiting for blood toclot is eliminated. The centrifugationperiod can be reduced considerably byincreasing the rotation speed.2. Higher yield. Approx. 15–20% moreplasma than serum can be obtainedfrom whole blood.3. Virtually no interference due to sub-sequent coagulation. Post-centrifugalcoagulation can occur in serum. Thiseffect does not occur in plasma.4. Results from plasma are more repre-sentative of the in vivo state comparedto serum (Fig. 13-1).5. Lower risk of haemolysis and throm-bocytolysis. In healthy persons, freehaemoglobin is about 10 times lessconcentrated in plasma than in serum.In plasma, the platelets remain intact invitro; there is hence no pseudohyper-kalemia here, as is found in serum(121).

What are the disadvantages ofplasma relative to serum?1. Protein electrophoresis is altered.Fibrinogen appears as a peak in the

region of the γ-globulins and can simu-late or mask an M-gradient.2. Method-dependent interference. Anti-coagulants can – as potential complex-ing agents and enzyme inhibitors –lead to method-dependent interference.Every new procedure should thereforebe tested for anticoagulant interference.3. Cation interference. When heparina-tes are used, lithium or ammonium mayinterfere with the methods for deter-mining them.

The annex summarises the present stateof knowledge on the use of anticoagu-lants in the individual analytes.

If serum or plasma separator tubes areused, renewed centrifugation followingstorage of the sample in the refrigeratorshould be avoided as this would leadto noticeable increases in the concen-tration of cellular constituents like potas-sium, inorganic phosphate and lactatedehydrogenase.

Types of plasma

Presently various different types of plas-ma are recommended for individualanalytes and test procedures. Thus buff-ered sodium citrate is recommended forcoagulation testing. Various types of plas-ma obtained from citrated blood areused:

Tab. 13- Different types of plasma

Plasma Relative centrifugal Centrifugationforce (g) time (min)

Platelet-rich 150–200 5Platelet-poor 1000–2000 10Platelet-free 2000–3000 15–30

K2-EDTA is recommended for haemato-logical cell analysis and analytes sensi-tive to metalloproteinase degradation.Heparin plasma is recommended fornearly all extracellular constituents.

2

When obtaining ablood sample, it is im-perative that attentionbe paid to ensuringthorough mixing of theblood with the anticoag-ulant; foaming shouldbe avoided.

Not more than 2 min-utes should elapse be-tween the beginning ofstasis and the mixing ofthe blood with anti-co-agulant.

Take a lavender tube!

34

AdditivesIn addition to anticoagulants such asEDTA, heparin, citrate and oxalate, otheradditives have been used for blood col-lection.

To insure that there is no confusion onthe part of the phlebotomist in identify-ing the proper tube, the stoppers andclosures of anticoagulant and additive-containing tubes are colour-coded.Thus, for anticoagulant-containing tubes,lavender is the stopper colour code forEDTA, green is for heparin and blue isfor citrate. Glycolytic inhibitors such asfluoride or iodoacetate either alone orin combination with an anticoagulantsuch as heparin or EDTA have beencolour-coded gray. Additional lettercodes have been included in the ISOnorm (89).

Fig.14-1 shows pictures of tubes usedin blood collection and identified bytheir proper colour code.

Tubes containing acid-citrate-dextrose(ACD A or B formulation) are used forthe preservation of cells and are colour-coded yellow.

HeparinHeparin, convenient for use in bloodcollection tubes, functions by accelerat-ing the inhibition of factor Xa by an-tithrombin III. In contrast to low molecu-lar weight heparin preparations, theconventional high molecular weightheparin used in blood collection tubesalso possesses antithrombin activity.

While the lithium salt of heparin is widelyused to obtain plasma for clinical chemi-cal analysis, blood collection tubes con-taining salts of sodium or ammonium he-parin are also commercially available.Ammonium heparin may limit the bloodurea nitrogen assay when ammoniumions are being measured (144).

EDTA

This compound functions as an anti-coagulant by binding calcium. EDTA isdiscussed in the haematology chapter (p. 54).

Citrate

Sodium citrate also functions as an anti-coagulant by chelating calcium. Sodiumcitrate is discussed in the coagulationchapter (p. 52).

Glycolytic inhibitors

Both sodium fluoride and lithiumiodoacetate have been used in bloodcollection tubes to preserve glucose.Mannose and fluoride have also beensuggested. When combined with ananticoagulant such as Na2-EDTA (1mg/mL of blood) or potassium oxalate(2 mg/mL of blood), the nominal con-centration of fluoride used to inhibitglycolysis is 2.5 mg/mL of blood (60mmol/L).

Fig.14-1Colour codes for anti-

coagulants andadditives described

by ISO 6710 (89)

Tab. 14- Additives of colour coded tubesTube Application Colour/letter code

1. Plain (no additive), Clinical chemistry and serology red/Zyields serum

2. Heparin (12–30 U/mL) Plasma chemistry green/LH or NH3. K2- or K3-EDTA Haematology and selected chemistry lavender/K2E or K3E

(1.2–2.0 mg/mL) determinations on plasma4. Sodium citrate Coagulation light blue/9NC

(0.105–0.129 mol/L)5. Sodium fluoride Glucose, lactate gray/FX

(2–4 mg/mL)/potassiumoxalate (1–3 mg/mL)

1

35

Additives and colour codes

Fluoride inhibits the enzyme enolase inthe glycolytic pathway and thereby pre-vents the degradation of glucose (34a).

In contrast to fluoride, iodoacetate actson glyceraldehyde-3-phosphate de-hydrogenase. After an initial loss ofglucose during the first 3 hours ofblood collection (an average loss of 9 mg/dL in healthy subjects), bothfluoride or iodoacetate are effective inpreserving glucose for at least 3 days(34a, 193). It should, however, be recog-nized that blood specimens with a highwhite blood cell, red blood cell orplatelet count will have more rapidglucose consumption, before inhibitorssuch as fluoride or iodoacetate becomeeffective (34a). In newborns, particular-ly because of the high haematocrit, asmuch as 68% of glucose can be con-sumed in blood specimens collectedwithout glycolytic inhibitors and storedat room temperature for 5 hours (126).To overcome this problem, inhibitorswhich inhibit the first enzyme hexoki-nase in the glycolytic pathway havebeen suggested. Such an inhibitor ismannose, which is used in combinationwith fluoride to better preserve glucose(117). The inhibition by mannose, whichis a competitive inhibitor, is short-lived,inhibiting glycolysis only up to 4 hoursafter blood collection. However, be-yond 3 hours of specimen collection,fluoride would have become effective;thus, a mixture of mannose (2 mg/mLof blood) and sodium fluoride (2mg/mL of blood) would have been ef-fective in minimizing the loss of glucosewhich would otherwise have occurredhad blood been collected using eithersodium fluoride or lithium iodoacetatealone.

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

Preservation of cells

Anticoagulant – additive mixtures suchas ACD (anticoagulant-citrate-dextroseor acid-citrate-dextrose) have beenused in blood collection tubes to pre-serve red blood cells. ACD is availablein 2 formulations, A and B. The differ-ence between the 2 formulations is theblood to additive mixture ratio.

Tab. 14-

The composition of ACD formulations

ACD A ACD BSodium citrate 2.2 g/dL 1.32 g/dLAnhyd. citric acid 0.73 g/dL 0.44 g/dLDextrose (glucose) 2.45 g/dL 1.47 g/dLACD/blood (v/v) 1/5.67 1/3pH 5.05 5.10

In the ACD A formulation, an additive toblood ratio of 1:5.67 is used whereasin the ACD B formulation the additive toblood ratio is 1:3.

ACD preserves red blood cells for 21days when blood is stored between 1and 6°C (22).

2

Fig.14-2Preservation ofglucose with glycolyticinhibitors (49)

100

90

80

70

60

500 2 4 20 22 24 (h)

rapid inhibiting mixture +NaF

storage time at room temperature

no inhibitor

gluc

ose

conc

entra

tion

(% o

f ini

tial)

NaF

Fax me a sample

36

Effects of time and temperature during transport

The transfer of samples to the laborato-ry can be accomplished in differentways. Normally, transfer times are shortwhen the laboratory is located close toor in clinics and represent no problem.The time from drawing the blood sampleto centrifugation, however, should notexceed one hour. Some analyticalprocedures require special additivessuch as sodium fluoride/ oxalate forquantification of lactate (9) or sodiumb-orate/serine EDTA for quantification ofammonia (56). Determination of freehaemoglobin in plasma requires gentlehandling of the EDTA blood sample.Transfer to the laboratory may proceedeither by courier or a pneumatic tubedelivery system. State-of-the-art systemsof the latter type ensure gentle transferthereby avoiding haemolysis. Suchsamples can be used to determinetarget analytes in clinical chemistry,

haematology or to perform blood gasanalysis (80). If for some technical reasona long distance transfer of the sample isrequired (e.g. by mail or laboratorycourier), then whole blood samplesshould be avoided. Fig.15-1 demon-strates the influence of temperature andduration of storage of clotted bloodsamples on some target analytes (84,163).

Release of potassium from erythrocytesis minimal at room temperature due tothe temperature-dependent activity of theNa+, K+-ATP-ase. This effect increasesboth at 4°C and above 30°C. Glucoseconcentrations decrease with increasingtemperature, whereas the opposite phe-nomenon is observed with inorganicphosphate because the activities ofphosphatases in serum and red bloodcells increase this compound. Asdemonstrated in Fig.15-1, duration ofstorage at a given temperature is aninfluencing factor. If a whole bloodsample is stored for two hours at 23°C,glucose concentrations decrease byabout 10 percent (Fig. 15-1).

Pathological samples may show devia-tions from the usually observed effectsof time and temperature. Time-depen-dent decrease of glucose in wholeblood samples is enhanced in leukocy-tosis. Similarly, the time-dependent in-crease in ammonia is enhanced in sam-ples with elevated γ-glutamyltransferaseactivities (56). Antibodies may alterhaematological cell counts dependingon the temperature dependence of theantibodies (see p. 78-79).

Many clinical chemical analytes suchas electrolytes, substrates or enzymesare not affected by a mail transporttime of up to four days. The haemoglo-bin concentration and the erythrocytecount are also stable. Major differences

Fig.15-1Temperature and time

effect of storage ofclotted blood without

anticoagulant onvarious serum ana-

lytes (163). ( ) 4 °C, ( ) 23 °C, ( ) 30 °C

mg/

L

glucose

chan

ge (%

)

1000900800700

500400300200

0 2 4 6 8 24 48

46

100

78

h

29

600

mm

ol/L

1110

9876543

0 2 4 6 8 24 48

100

252

198

chan

ge (%

)

h

potassium


37

Effects of time and temperature during transport

are observed for the haematocrit, themean cell volume and bilirubin (Fig. 15-2). A reliable examination of a differen-tial leukocyte count requires prepara-tion of the blood smear within threehours of sampling. The included Annex”The Quality of Diagnostic Samples“may be used to find additional detailson analyte stability in full blood andserum/plasma.

Numerous studies related to the stabi-lity of clinical chemical analytes existcompared to the few investigations onthe stability of haemostasiological para-meters (83, 216). As shown by Heil etal. the stability of coagulation factors inpatient samples is dependent onwhether they are on heparin therapy ornot. For the thrombin time this is clearlydemonstrated in figure 15-3. The caus-es for the changes of stability aremanyfold, i.e. loss of the haemoglobinbuffering system after plasma separa-tion from the red blood cells, an in vitrofibrinolytic effect, or drug influences onplatelets. The results of the study (83)can be summarised as follows: citratedblood samples of patients without he-parin therapy for the determination of

prothrombin time, APTT, thrombin time,protein C and factor V are stable foreight hours at room temperature, pro-vided that the plasma is not separatedfrom the erythrocytes. This is not validfor factor VIII and protein S. The samplestability of patients with heparin thera-py, stored at room temperature or inthe refrigerator is below eight hours forthe global tests and the determinationof single coagulation factors. There-fore, all patient samples should beanalysed within four hours at room tem-perature after sampling. If this is notpossible, platelet poor plasma shouldbe stored at –20° C.

chan

ges

(%)sodium

potassium

calcium 6

4

2

0

-2

-4

-6

-8

-10

albuminbilirubin

creatininealkaline phosphatase

alanine aminotransferase

aspartateaminotransferase

γ-glutamyl-transferase

lactatedehydrogenase

haematocrit

mean cell volumeleucocytes

haemoglobinerthrocytes

Fig.15-2Stability of various analytes during mailtransport (13, 122)

Fig. 15-3Thrombin time (TT) de-termination in plasmasamples of patientswith and without he-parin therapy, stored atroom temperature (RT)and 6° C (83)( ) without heparin at RT

( ) with heparin at RT( ) without heparin at6° C( ) with heparin at 6° C

120

110

100

90

80

70

60

0 24 48 72 96 168

Abw

eich

ung

vom

Aus

gang

swer

t (%

)

508 h

Samples in transit

38

When blood or other body fluids fromhuman subjects are mailed to a distantlaboratory, several safety regulationshave to be complied with. In addition,the integrity of the sample has to be pre-served to insure accurate analysis bythe investigator. Specimens that aremailed must “withstand leakage of con-tents, shocks and pressure changes,and other conditions incident to ordinaryhandling in transportation” (50, 159,196).

The persons dispatching diagnosticsamples have to assure, that the con-tents are packaged in a way insuringarrival in an undisturbed state. No riskshould occur for humans, animals andthe environment during transport.

Regulations concerning transport bypost are reported in national standards(43). Here samples with infectious sub-stances have to be treated differentlycompared to materials with a low riskof infection (like most samples of blood,serum, urine, stool, swabs, slides andfilter papers). For posting of diagnosticsamples even if non volatile the pack-age can be sent by letter post. Pack-ages with infectious materials have tobe labelled with the remark: DIAGNOS-TIC SAMPLE/INFECTIOUS HAZARD. Forinternational traffic and posting the description in French language:”Matières Biologiques Périsablés” is

required. The responsibility for the post-ing of infectious materials is with thedispatching physician’s site.

In Europe the standard package EN829 (Fig. 16-1) is accredited (50). Noglass is allowed as sample material toreduce the risk of breakage and possi-ble harm to people involved in trans-port.

The package for biological materialsconsists of the following parts:

● The inner package for the samplematerial,

● absorbing material,● the outer package ensuring speci-

men records and laboratory forms,● the box or mailing bag.

Instead of the outer package severalspecimen containers up to 500 mL maybe packaged in one box consisting ofcard board, wood, suitable plastic ormetal according to the regulations forbiohazard transport.

Remarks: In any case where infectioussubstances are contained in the pack-age, additional secondary container isneeded to prevent any leak of materialby any mechanical challenge.

Fig. 16-1Biohazard Label for

air-shipped specimens

Fig.16-2Infectious substance

label for packagecontaining etiologic

agents

A biohazard label as

shown in Figure 16-1

should be affixed to the

package.

▲

ETIOLOGIC AGENTS

IN CASE OF DAMAGEOR LEAKAGE, NOTIFY

CENTERS FOR DISEASE CONTROL(404) 633-5313

BIOHAZARDPackaged in Complance with 42 CFR Part 72

INFECTIOUS SUBSTANCEIN CASE OF DAMAGE OR LEAKAGE

IMMEDIATELY NOTIFYPUBLIC HEALTH AUTHORITY

IN U.S.A.NOTIFY DIRECTION – ODC

ATLANTA GA404/633-5313

6

39

Legal standardization for mailing samples

Always remove injection needles whenmailing blood sampling systems.

Package glass slides adequately to en-sure they do not get damaged ifknocked, dropped or if pressure is ap-plied.

Ship stool specimens in leak-proof,screw-capped containers.

When mailing dried blood specimenson filter paper, place in a strong paperenvelope and then seal in plastic-lined,padded post bag. This provides protec-tion against potentially infectious driedblood specimens and ensures the inte-grity of the specimens during transport.

For shipping frozen and refrigeratedspecimens, an insulating material suchas a polystyrene container is adequate.Dry ice should be used for freezing.

Caution should be taken to insure thatthe container packed with dry ice isable to release carbon dioxide gas soas to avoid a build up of pressure thatcould cause the package to explode.

NCCLS document H18-A2 describesprocedures for the handling and trans-port of diagnostic specimens and etio-logic agents (159).

In Europe details are regulated by Eu-ropean Standard prEN 829 for in vitrodiagnostic systems (50). Here detailed

information is found together with therequirements and testing procedures re-garding transport packages, samples,absorbing materials and protective ves-sels. The label to be used is shown inFig. 16-4.

Fig. 16-3Packaging of speci-mens for transportaccording to NCCLS(159)

primary container

absorbent packingmaterial

cap

secondary container

specimen record(CDC 3.202)cap

EA label

shipping container

address label

waterproof tape

culture

absorbentpackingmaterial

cross-section ofproper packing

Fig. 16-4Label to be used fortransport of medicaland biological speci-men according to EN 829 (Germanversion)

Medizinisches Untersuchungsgut Biologisches Untersuchungsgut

How to keep a sample ”fresh“

40

10 rules and somerecommendations

1. The procedure is governed by thestability of the constituents of the sample.The most important causes for altera-tions to the quality of specimen are:

● Metabolism of the blood cells● Evaporation/sublimation● Chemical reactions● Microbiological decomposition● Osmotic processes● Effect of light● Gas diffusion

2. Rapid transport and short storagetimes improve the reliability of labora-tory results.

3. Specimens and samples are preservedlonger the cooler they are stored (butnote exceptions!).

4. Specimens and samples shouldalways be stored in closed vessels(evaporation!).

5. The danger of evaporation also ex-ists in refrigerators (condensation ofmoisture on the cooling elements).

6. Storage problems are reduced if disposable sampling systems are used.

7. Separating agents (e.g. gel sepa-rators) improve the serum/plasmayields and enable serum to be left in the original tubes above the blood (111).

8. Avoid shaking the sample vessels(pneumatic tube dispatch systems!): riskof haemolysis.

9. Always store sample vessels con-taining blood vertically; the clottingprocedure is accelerated.

10. Label infectious material and handleit with particular care.

8 special rules and some moreuseful recommendations

1. Avoid storing of whole blood. Infor-mation on sensitive analytes is given inthe Annex (see enclosure).

Blood samples should reach the labora-tory within 45 min of collection in orderto ensure that centrifugation and sepa-ration of the sample is carried out with-in 1 hour (49, 112, 159).

2. Avoid glycolysis to keep glucose,lactate and pH stable. Glycolysis canbe avoided by the addition of an in-hibitor in conjunction with an anticoag-ulant (171, 220).

3. Avoid the effect of light otherwisethere will be a fall in the values ofbilirubin, vitamin C, porphyrins, creatinekinase (CK) and folic acid.

4. Reduce contact with air as far aspossible. If this is not done, evaporation/sublimation will result in an apparentincrease in the concentration/activity

calc

ium

mm

ol/L

2.0

1.5

1.0

0.5

upper middle lower

upper

middle

lower

Fig.17-1Formation of concen-

tration gradients ofcalcium in plasma

samples after rethaw-ing without mixing

41

Storage of samples in the laboratory

of all non-volatile components. This isparticularly the case when the volumeof the sample is relatively small and thesurface area is relatively large.

5. Whole blood should not be stored inthe refrigerator. When urine is cooled,salts may precipitate out of the solution(calcium and magnesium phosphate,uric acid).

6. For certain analytes, the specimens/samples should not be deep frozen.Failure to observe this can result indeviating results for the followinganalytes:

Tab. 17- Examples of blood and urine constituents which should not bestored frozen

Sample Analytes

Serum/plasma: Lipoprotein electrophoresisApolipoprotein A-I and BLDL-cholesterol (prevented bythe addition of glycerol)Fibrin monomer positive plasma*

EDTA-Blood HaematologyUrine IgG

SedimentUric acid (precipitations!)

*Negative test result, prolonged PTT, shortened thrombin time, shortenedreptilase time (215).

7. Correct thawingA very common source of error is theinadequate mixing of deep-frozensamples after they have been thawed.Concentration gradients are producedduring thawing as the concentratedsolution first melts and then runs downthe sides of the vessel (see Fig. 17-1).

After thawing, the sample should there-fore be inverted several times, avoidingthe formation of foam. Look for

1

undissolved material and, if necessary,bring into solution by careful warming.

8. Store samples after analysis in sucha way as to permit the confirming ofresults, checking the identity of samplesor performing additional tests formedical or legal reasons:

Tab. 17- Recommended storage time andconditions for analytical samples

Samples for Storage time Temperature

Clinical Chemistry: 1 week RefrigeratorImmunology: 1 week RefrigeratorHaematology: 2 days Room temperatureCoagulation: 1 day RefrigeratorToxicology: 6 weeks RefrigeratorBlood grouping: 1 week Refrigerator

(at least)

2

Fig.17-2Storage of samplesshould enable easyreidentification forconfirmatory tests

What has to be done on specimen arrival?

42

Centrifugation

Centrifugation of clotted blood to ob-tain serum should be performed aftermaking sure that the blood has clotted.Normally, the waiting time for blood toclot is approximately 30 min. However,patients who are on anticoagulant ther-apy, or those with coagulation defectswill have delayed clotting.

Centrifugation of clotted blood to ob-tain serum or anticoagulated blood toobtain plasma is typically performed at1000 to 1200 g for 10 to 15 min. Forthe production of platelet-free plasma acentrifugation for 15 – 30 min at 2000 –3000 g is necessary (see Tab. 13-2). Incoagulation procedures, citrated wholeblood should be centrifuged at 2000 gfor 15 min (159).

The relative centrifugal force can becalculated either by using an empiricalequation or by using a nomograph asshown in Fig. 18-1. The equation used

to calculate the relative centrifugalforce (rcf) is as follows:

rcf = 1.118 x 10-5 x r x n2

Where r is the mean radial distancefrom the axis of rotation in centimetersand n is the speed of rotation in revolu-tions per minute (rpm). 1.118 x 10-5 isa constant which is derived from thecentrifugal force as multiple of g.

Centrifugation is usually performed bet-ween 20 to 22°C. However, analytesthat are labile during centrifugation atambient temperature, especially if thetemperature increases during centrifu-gation, should be centrifuged at refrig-erated temperature (4°C). However, re-frigeration can lead to leakage ofpotassium from the cell, thus spuriouslyincreasing its value (see Fig. 15-1).

Specimens should not be recentrifugedafter sampling the serum or plasma. Indoing this the ratio of plasma water tocell volume may be altered thus caus-ing alterations in analyte concentra-tions. Specimens with a gel barrier ma-terial should never be recentrifuged.

The time and centrifugal force appliedto the sediment of heparinized bloodshould be such as to leave no plateletsin the plasma layer. Failure to com-pletely sediment platelets will result inspurious increases in potassium, lactatedehydrogenase, acid phosphatase andinorganic phosphate from platelets re-maining in the plasma (Fig. 18-2) of thesample (69). Fig. 18-3 shows the visualcontrol of plasma after centrifugation atvarious speeds.

Microcollection tubes with or withoutanticoagulants can be centrifuged toobtain either serum or plasma in a mi-crocentrifuge with an adapter that ac-

Fig. 18-1Nomograph for

calculation of relativecentrifugal force

25

20181614121098765

4

2

3

10.0005.0002.0001.000

500200100502010

20.00030.000

20.00015.000

10.000

6.000

4.0003.000

2.0001.5001.000

50

20relativecentrifugalforce

3

rotating radius

speedcm

g

revolutionsper minute


43

Specimen processing, centrifugation, distribution

cepts such tubes. The centrifugation ofsuch microcollection tubes is performedat speeds ranging from 6,000 to15,000g for a minimum of 90 sec.

A preventive maintenance program forcentrifuges should include a schedulefor periodic checking of centrifugationspeeds attained at a specified speedsettings using a tachometer.

Sample handling after centrifugation

After centrifugation, samples may betransferred directly to the analyzer.Ideally, the analyzer needle takes theanalytical sample by piercing theclosed stopper after the sample has beenmixed. In most laboratories, however,the stopper has to be removed and thesamples distributed. To prevent evapo-ration, this should be done shortly beforeanalysis. Subsamples should follow thesame rules as the primary specimensregarding identification, storage condi-tions and safety aspects.

To avoid contact with blood containingother potentially infectious materials,subsampling should be avoided as faras possible.

This is facilitated by the use of separa-tors in tubes. Alternatively, distributioncan be performed by mechanical de-vices (Fig. 19-1, p. 44).

Fig. 18-2Effect of plateletcontamination due toinsufficient time andcentrifugal forceapplied to heparinizedblood

Fig. 18-3Visual control ofadequacy of centrifu-gation before analysisof serum

platelet-freeplasmalayer

platelet sediment

cells

plateletgradientin plasma

platelets

cells

Insufficient time and centri-fugal force. Sample probewill pick-up platelets presentin plasma giving rise tospurious results.

Sufficienttime andcentrifugalforce.

Continuous or batchwise?

44

Workflow can be defined as the stepscarried out from the time a specimenarrives in the laboratory to the time theresults are reported to the physician.This workflow then determines the turn-around time (TAT) of laboratory results.A broader definition of workflow wouldencompass steps from the time thephysician orders a test to the time theresults reach him.

The importance of preanalytical time interms of total TAT can be appreciatedfrom a study by Godolphin which isdetailed below (62).

Tab. 19- Contribution of the preanalytical phaseto total turnaround time (TAT)

% of TATPreanalytical 57.3%Analytical 25.1%Postanalytical 17.6%

In recent years, automated samplesorter systems have been introduced tocope up with the workload of largereference laboratories and automatedaliquoting systems have provided a

1

simplified alternative to time-consumingand cumbersome manual aliquotingprocedures. Fig.19-1 illustrates an auto-mated sample sorter system (127).

Consolidation of work areas such asroutine, special chemistry and haema-tology sections should facilitate work-flow. Efficient workflow is dependenton streamlining sample processing, ali-quoting and distribution steps (61).

Discontinuous steps such as centrifu-gation and labeling samples adverselyaffect total turnaround time.

Robotics

Robots are devices that can be pro-grammed to perform specific tasks. Assuch, robotics is a term used to describethe utilization of robots to performspecified repetitive mechanical tasksthat are programmed and hence underelectronic and computer control.

Robots of varying flexibility are avail-able (139). Robots with three degreesof freedom that can move about in athree-dimensional space, but are inca-pable of rotation are called Cartesianrobots and have applications rangingfrom sampling devices on automatedanalyzers to pipetting stations for han-dling liquids (54).

Robots with four degrees of freedomare called cylindrical robots and are ca-pable of moving in and out of planewith a wrist roll. By the use of roboticarms, these cylindrical robots have beenused for sample preparation tasks suchas in blood typing or in multiple stepssuch as sample extraction, separationof aqueous and organic phases andsample injection associated with highperformance liquid chromatographicprocedures.

Fig. 19-1Automated sample

sorter system(with kind permissionof P. Mountain, Auto-lab, Toronto, Canada)

45

Preanalytical workflow and robotics

Highly flexible robots with five degreesof freedom are called jointed robotsand are very versatile with their wristrotatory motion and ability to reach theremote positions on an instrument.Fig.19-2 illustrates robots of varying de-grees of flexibility.

Robots are also used to transport speci-mens to the laboratory across tapedtracks (118).

M. Sasaki, a pioneer in the use ofrobotics, used a conveyer belt systemto transport samples to the roboticanalyzers which in turn were con-nected to the conveyer belt (Fig.19-3).Sasaki has used robotic analyzers toperform a variety of tests that includeserological aggregation tests includingAIDS tests, blood transfusion tests suchas ABO blood typing, cross-matching,Rh factor testing and hormone analysis(181, 182).

In the meantime different systems of ad-vanced analytical systems including ro-botic regulation have been developedand incorporated into working labora-tories (85, 86, 207).

While robotics can contribute to theefficiency of workflow, by their verynature they require special considera-tions. This is due to the fact that themovements of robotic arms are underthe control of complex electronic circu-its. As such, any fluctuation in line volt-age can disrupt the operation of the ro-bot. Hence, having access to anuninterrupted filtered power supplywith back-up batteries is essential forrobotic operations.

Finally, cost and space considerationshave to be considered in the process ofdetermining the suitability of roboticoperations in a clinical laboratory.

Fig. 19-2Robots of varyingdegrees of flexibility

Fig. 19-3Conveyer belt systemto transport samplesto robotic analyzers(with kind permissionof M. Sasaki, Kochi,Japan)

▲

Cartesian robot

cylindrical robot

jointed robot

Safety aspects during the preanalytical phase

46

Steps to ensure safety are paramount tothe protection of the health care worker.The NCCLS document GP17-T providestentative guidelines on clinical labora-tory safety (154). This document outlinessteps for the maintenance and inspectionof the laboratory, the general require-ments for personal and laboratorysafety, warning signs and labels needed,fire prevention and control, electricaland radiation safety, handling of com-pressed gases and carcinogens, chemicaland microbiological hazards andhazardous waste disposal (154).

Specifically, the disposal of specimens,needles, tubes, and chemicals arediscussed in this chapter.

Disposal of needles and other sharp objects

The disposal of all sharp objects suchas needles is accomplished by placingthem in leak-proof, puncture-resistantcontainers with appropriate labels andcolor coding, an example of which isillustrated in Fig. 20-1.

Fig. 20-1Disposal of needle into

sharps container

a) Sampling with a safety needle

c) Single use holder: immediately after sampling, discard the needle holder combination into a sharpscontainer.

b) Quick release holder; discard the needle intosharps container by pressing the release button. Theholder then can be reset ready for reuse.

a)

b)

c)

BIOH

AZAR

D W

ASTE

SING

LE U

SE O

NLY

SHARPS DISPOSALSINGLE USE ONLY

FILL TO THIS LEVEL ONLY

Needle Disposal Container

FORCING OR OVERFILLING SHARPS INTO CONTAINERMAY CAUSE SERIOUS INJURY

WARNING: This container is puncture-resistant, but notpuncture-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.

d) Container

d)

47

Disposal of specimens, needles, tubes and chemicals

Reshielding of sharps and the bendingand breaking of needles should beavoided.

However, should recapping becomenecessary, either a one-handed scoopor, preferably, a reshielding deviceshould be used.

Simple reshielding devices are available,which allow the phlebotomist to reshieldthe needle with minimal risk of needleprick injury (Fig.20-2).

Specific safety devices have also beendeveloped for the disposal of microblood collection sets (Fig. 20-3).

Tube and sample disposal

Specimen collection tubes containingblood and which are intended for dis-posal should be placed in biohazardbags that can withstand autoclaving.These bags should be placed in leak-proof containers and then tightly closed.

Bulk body fluids such as urine, vomit,feces and other body fluids may bedisposed of by flushing down the toilet.

Containers of fluids such as blood bagsshould be incinerated after placing inbiohazard waste containers.

Chemicals

Chemical waste can be ignitable,corrosive, reactive and toxic (152).

Examples of ignitable chemical wasteinclude volatile flammable liquids suchas organic solvents (e.g., alcohols, ace-tone, xylene, toluene, etc.), oxidizerssuch as peroxides and nitrate salts andflammable gases such as butane, silaneand hydrogen. These materials should belabeled with the appropriate hazardouschemicals label shown in Fig. 20-4.

Disposal of flammable solvents via asink or flush toilet is not recommended.If such solvents are readily dissolvablein water, they could, in small amounts,be disposed of by pouring down a sink,

Fig. 20-2Safety needle forarterial collection andnestable sharpscontainer

5 min

3

2

Immediately after sampling, activate needle safety mechanism and discard the needle into sharps container.

Safety aspects during the preanalytical phase

48

followed by copious amounts of water.It is best to collect flammable solvents insafety cans or drums and store in astorage cabinet prior to collection by adisposal collector or company. Ether andchlorinated solvents should be collectedin separate cans. Other solvents maybe combined in one can.

Examples of corrosive solvents are strongacids such as sulfuric, hydrochloric,and phosphoric acid and bases such as ammonia (ammonium hydroxide).

Toxic, corrosive and inflammablechemicals should not be used as preser-vatives in the preanalytical phase.

Never add urine to concentrated acids.Such acids should be diluted byadding them slowly down the sides ofthe urine container. Disposal of strongacids and corrosive materials shouldpreferably be done by pouring downthe sink, followed by copious amountsof running cold water from a tap.

Thus when urine is collected in hy-drochloric acid, the first portion shouldbe collected before acid is added.

Toxic chemical waste such as toxicmetals can pose a threat to ground water.

Fig. 20-3SAFETY LOK™ Systemfor reshielding needleof blood collection set

a) When sampling is complete apply gauze pad onpuncture site. Grasp the yellow shield between yourthumb and forefinger while using your remaining fin-gers to hold the tubing against the palm of your hand.

b) With the tubing held taut, advance your thumband forefinger to slide the safety shield forward untilan audible “click” is heard.

c) The click confirms that the shield is locked into pla-ce, covering the needle. Dispose the blood collectionset in a suitable container.

a)

b)

c)

49

Disposal of specimens, needles, tubes and chemicals

The Environmental Protection Agency inthe United States of America (EPA) listschemicals that constitute toxic waste(222). A European list of maximal allow-able concentrations of chemicals in air,water and foodstuffs is also available (42).

Finally, to be knowledgeable aboutchemical hazards, each laboratory shouldhave a file of material safety datasheets (MSDS) that list the hazardousproperties of each chemical, and whichcan be readily consulted in cases ofemergency or when there is doubt as totheir hazardous nature.

Fig. 20-4Safety labels of United States andEuropean origin

FLAMMABLE

D A N G E R

P E L I G R O

INFLAMMABLE

DANGER

HAZARDOUS WASTESTORAGE AREA

CORROSIVE

irritant corrosive harmful to theenvironment

explosive oxidizing flammable toxic

What is needed before blood transfusion?

50

Ensuring patient and sample identityWhile processing blood and bloodproducts 41 % of the reported defectswere shown to be related to the prean-alytical phase, 55 % to the postanalyti-cal phase and only 4 % to the analyti-cal phase according to a study ofBoone et al. (20). The group at highestrisk are patients issuing 10 or moreunits per day.

Most haemolytic transfusion reactionsresult from discrepancies in patient orsample identity. The frequency of ad-ministration of a wrong transfusion bymeans of mismatching the samples is1:60000 transfusions. Therefore, requestforms for blood transfusion must con-tain the first name, last name, the dateof birth and, if possible, an identificationnumber unique to the patient. Before blood sampling is performed,active control of patient identity has tobe thoroughly ascertained by askingthe subject for his or her last name, firstname and date of birth. Blood samplesmust be collected in correctly labeledtubes. The name of the person who hasdrawn the sample must also be docu-mented.

The right sample

● Serum is used for immuno-haemato-logical tests. Anticoagulants such asEDTA or citrate prevent complementactivation. Consequently, complement-activating antibodies are not detectablein such plasma samples.● The use of haemolytic samples isnormally not allowed because antibody-induced haemolysis can be masked.● The patient sample for pretransfusiontesting should not be taken less than 72hours prior to the test.● Each patient sample for immuno-haematological examination must bestored at 4–6°C for one week follow-ing the test.

● For cold agglutinin determination, theblood sample must be kept at 37°C un-til serum has separated.● Citrate blood is necessary if identifi-cation of erythrocyte-bound antibodiesrequires elution.● EDTA blood samples are used for thedetection of in-vivo complement bind-ing by erythrocytes. For special erythro-cyte antigen testing, citrate-anticoagu-lated blood should be used.● Samples of blood contaminated withWharton‘s jelly may agglutinate spon-taneously. The cells should be separatedby washing three times with 0.9% so-dium chloride solution.● Interferences of the agglutinationreaction involve either pseudo- orpolyagglutination. Pseudoagglutination(rouleaux) can be caused endogenous-ly or exogeneously (Fig. 21-1).● Exogenous factors are infusions ofdextran, polyvinyl-pyrolidon or fibrino-gen; among endogenous variables aredisproteinemias in immunocytoma, livercirrhosis or hyperfibrinogenaemia.● Polyagglutination is a condition inwhich red blood cells are agglutinatedby a high proportion of group-compati-ble sera. The same phenomenon occur-ring in vitro is called polyagglutination(28). This can occur during viraemia orbacteriaemia (endogenously) or afterexogenous bacterial contamination ofpatient erythrocytes. Bacterial enzymesmodifiying erythrocyte antigens releas-ed are one of the causes of in vitro andin vivo polyagglutination. T-polyaggluti-nation is caused by a microbial neu-raminidase, the TK-polyagglutinationby a β-galactosidase or the acquired B-antigen by deacetylase.Non-microbial polyagglutinations arealso known. These are caused by somaticmutation of a pluripotent haemopoeticstem cell (Tn) or congenital geneticdefects.


51

Special aspects in immune haematology

● All immuno-haematological resultsshould be observed and interpreted bytwo independent persons who shouldsign for the correctness of the proce-dure.

Storage of blood for transfusion

Blood units have to be stored in specialrefrigerators equipped with an auto-matic temperature recording systemand an optical or audible alarm system.The low alarm activation should be setat 3.5 ± 0.5°C and the high alarmactivation at 5.5 ± 0.5°C. The surfaceof the blood container must be cleanand dry. Any item that could result inthe puncture of blood containers shouldbe removed from the storage area.Products for transfusion must be storedseparately from reagents or used pilottubes. The storage refrigerator shouldbe kept clean. A standard procedurefor cleaning the storage refrigerator ismandatory (5).

What has to be done beforetransfusion?Both on delivery of the blood unit andprior to transfusion, all information onthe container and the accompanyingtransfusion form have to be checked foridentity. The ABO-status of the donormust be evaluated immediately beforetransfusion by means of a bedside test.In the case of autologous transfusion,ABO status has to be checked for boththe blood unit and the recipient by bed-side testing. The residual blood of thetransfused unit has to be kept for atleast 24 hours after infusion at 2–8°C(230).

Fig.21-1Pseudoagglutination(rouleaux)

Fig. 21-2Blood typing by gelfiltration test

Why a separate tube for the coagulation test?

52

Specimen collectionThe results of coagulation screeningtests such as the prothrombin time (PT,Quick Test) and the activated partialthromboplastin time (APTT) are decisive-ly influenced both by the anticoagulantused, its concentration and ratio toblood, and by the manner of collectionand further processing of the specimen.Citrate can be regarded as standardanticoagulant (105).

Concentration of the anticoagulant

Prothrombin time (PT) reported as theinternational normalised ratio (INR) issensitive to the concentration of citrate.INR values, especially with a respon-sive PT reagent similar to the WHOthromboplastin, with an internationalsensitivity index (ISI) equal to 1, aregenerally higher with a 0.129 mol/L(3.8 %) than with a 0.109 mol/L (3.2 %)sodium citrate (2, 142). The differencesin INR between the two concentrationsof citrate can vary from 0.7 to 2.7 INRunits (2).

Hence a laboratory should not inter-change the two concentrations of cit-rate when PT is reported in INR units onpatients who are undergoing oral anti-coagulant therapy.

In addition to maintaining the nominalanticoagulant to blood ratio (1+9), the

amount of headspace above the bloodis also a variable that can affect theAPTT result (2, 194). Apparently, the in-crease in surface area in a tube fa-vours platelet activation with the subse-quent release of platelet factor 4,which in turn neutralises some of theheparin causing a shortening of theAPTT value (2, 194).

The recommended citrate concentrationis 0.105 mol/L (3.2 %) (89, 155). A cit-rate solution buffered to pH 5.5 ispreferable to an unbuffered solution asin the buffered solution the pH of thespecimen is closer to the physiologicalrange (146). If the haematocrit isabove 0.55 a correction is necessaryeven if the ratio of anticoagulant toblood is 1+9 (1:10). This is because theplasma compartment is reduced, result-ing in an excess of citrate in the speci-men container which in turn complexeswith the calcium ions added duringmeasurement of the PT and APTT. Thusthe clotting time is prolonged.

Specimen processing

After carefully inverting the specimencontainer several times to exclude clotformation and checking that the ratio ofanticoagulant to blood is correct, theclosed specimen tube is centrifuged at2000 g for 10 minutes (155). Specimenswith invisible clotting or haemolysisshould not be used as activation of clot-ting factors may have taken place. Inaddition, it is very important to look outfor lipaemic or icteric specimens asthese can cause interference in thecase of photo-optical measurements.

Unopened specimens for the perform-ance of coagulation screening tests (PTand APTT) should be transported to the laboratory at room temperature(22–24°C) but not on ice.

Fig. 22-1Effect of haematocrit on the APTT using a

buffered citrate solution(0.129 mol/L)

80

60

40

20

0.3 0.6 0.9

APT

T (s

ec)

haematocrit (fraction)

Strict observation of aratio of 1+9 (citrate

solution to blood) fordetermination of the

APTT and other coagu-lation screening tests is

recommended.

▼

If several evacuatedblood collection tubes

are used, the specimenfor coagulation testingshould be collected in

the second or third tubein order to minimisecontamination with

tissue thromboplastin.

53

Special aspects in immune haematology

within two weeks. In specimens frozenat –70°C the factor VIII activity is stablefor up to one year.

Evaluation of fibrinolysis and monitoring of fibrinolytic therapyAfter a freezing and thawing process,identical results are only obtained if theplasma contained no fibrin monomers,fibrin degradation products or heparinbefore freezing.In plasmas which contain fibrin mono-mers gelling processes after thawingcan result in negative fibrin monomerdetection, prolongation of the APTT andprolonged thrombin and reptilase times.In plasmas with considerably prolongedthrombin times (heparin, FDP) instabilityafter thawing can lead to shortening oreven normalisation of the thrombin timeand APTT after thawing (216).Specimens for monitoring fibrinolytictherapy with streptokinase or uroki-nase, for example, should be collectedinto tubes containing a mixture of EDTAand aprotinin as this combination im-mediately inhibits the streptokinase orurokinase induced plasmin activation(146). For this, aprotinin in a concen-tration of 150 kIU/mL blood should becombined with trisodium citrate (10mmol/L) or EDTA (4.2 mmol/L).Unlike FDP measurement, which re-quires a tube containing 10 units ofthrombin and 1835 units of soybeantrypsin inhibitor/mL of blood detection,D-dimer, which reflects the fibrinolyticactivity, can be performed in citratedplasma.

The activity of rt-PA (recombinant tissueplasminogen activator) in the bloodcan be effectively inhibited with a mix-ture of 5 mmol/L D-phenylalanine-pro-line-arginine-chloromethylketone and10 mmol/L citrate or 4.2 mmol/L EDTA(138, 142).

The table in the Annex gives details onspecimen stability for the different co-agulation tests.

The following general principles canserve as a guide.● If the test is to be performed immedi-ately the specimen is kept at room tem-perature. The plasma can be left standingon top of the packed cells after cen-trifugation.● The collection vessel should be closedto avoid changes in pH due to evapo-ration of volatile carbonic acids.● Exposure to high temperatures (in-cluding direct sunlight) must be avoid-ed at all costs.● Specimens should not be refrigerated(+2 to 8°C) as cold activation of factorVII, and also of factors XI and XII, canlead to shortened clotting times in therespective screening tests.● For tests to be performed at a laterdate platelet-free (< 5000/µL) citratedplasma is aliquoted and frozen inclosed tubes.● Frozen specimens should be thawedquickly in a water bath at +37°C andmixed thoroughly, making sure that anycryoprecipitates are completely dis-solved. Repeated freezing and thawingis not recommendable.

Specimen storage

The plasma should be left standing onthe sedimented cells and used within 2to 6 hours (for details see Annex). If thestorage period is to exceed 4 hours theplasma specimens can be kept in therefrigerator for 4 weeks at –20°C, andwith rapid freezing to –70°C the stor-age time can be extended to 6 months(83, 155).

Factors V and VIII are unstable. The fac-tor VIII activity (VIII.C) of the samplesfrozen at –20°C should be measured

The measurements ofthe PT and APTT areleast influenced if theplasma is analysed atroom temperature within2 hours.

For single factor assays,

particularly of factor

VIII, the blood should be

cryocentrifuged (+4°C)

as soon as possible

after collection and the

plasma frozen at –20°C

to –70°C.

Blood cells are sensitive!

54

Optimum anticoagulantThe International Council for Standard-ization in Haematology (ICSH) has rec-ommended dipotassium EDTA (K2-EDTA)(ethylenediaminetetraacetic acid) asthe anticoagulant of choice for the col-lection of blood specimens intended forblood cell counting and sizing (170).

K2-EDTA was selected in preference toNa2-EDTA because of the greater solu-bility of the potassium salt compared tothe sodium salt.

With all EDTA salts, the cells shrink, thusaffecting the centrifuged but not the cal-culated haematocrit. Because of thelower pH of Na2- and K2-EDTA com-pared to K3-EDTA, the cells swell, thuscompensating for osmotically inducedcell shrinkage. Calibration of electronicblood cell counters for mean corpuscu-lar volume (MCV) using the micro-haematocrit value obtained from bloodspecimens collected in either Na2- or K2-EDTA have been reported to give ac-ceptable results, in contrast to the unac-ceptable results obtained when micro-haematocrit values obtained from bloodspecimens collected in K3-EDTA wereused to assay commercially availablecontrol material (170, 188).

With a variety of techniques now incor-porated in the new automated instru-ments for counting, sizing and provid-ing a 5-part white blood cell differentialcount, the question has been askedwhether any salt of EDTA is the adequateanticoagulant for haematology (169).

Platelets change from their discoid to aspherical shape upon blood collectionin EDTA, thus introducing an error in themean platelet volume (MPV) determina-tion.The influence of EDTA on the stability ofwhite blood cell populations, lympho-

cytes being relatively more stable andneutrophils and monocytes more likelyto be influenced when stored in EDTA,introduces a variable that is com-pounded by the variation in the clusteranalysis software packages used bythe different analyzers.

Ratio of anticoagulant to blood

The volume of blood drawn should ensuremaintenance of the recommendedconcentration range for the EDTA salt of1.2–2.0 mg/mL (180).

Since the concentration of EDTA has aneffect on neutrophil morphology, thequality of the peripheral blood smearcan be compromised if attention is notpaid to the anticoagulant to blood ratioand the time elapsed between bloodcollection and preparation of the smear.

At concentrations of EDTA of up to 1.50g/L of blood in blood specimens notover 1 hour old, minor changes appearin neutrophil morphology. As the con-centration of EDTA increases, more seri-ous changes such as loss of bridges be-tween lobules, loss of cytoplasmicboundary and early cross-over occurswithin the first hour.

The effect of increase in the concentrationof EDTA over the nominal value decreasesthe centrifuged microhaematocrit value,the decrease being more pronouncedwith K3-EDTA than with K2-EDTA.

However, with automated instruments,the mean corpuscular volume was notinfluenced at K3-EDTA concentrationsup to 10 times the nominal value; andresults obtained with K2-EDTA wereshown to be dependent on the instru-ment used (63).

55

Special aspects in haematological analysis

Using the recommended concentrationof EDTA salts (K2- and K3-EDTA) and per-forming analysis between 1 and 4hours after blood collection, no signifi-cant difference was seen in resultsobtained with blood collected in eitherof the 2 anticoagulants (63).

Specimen collection and handling

Thorough mixing of the blood specimenwith the anticoagulant by inverting thetube several times is a prerequisite. Thetype of mixer (rocking versus rotary)used may affect the extent of mixing,especially if the tube is overfilled, thusyielding inaccurate results (166).

Transport, storage and stability of analytesBecause of the variations betweennewer automated instruments, includingreagents, it has been recommendedthat the EDTA anticoagulated blood beanalyzed within 6 hours of collection(150, 170). In some cases, however,this time is already too long to ensureconstant results. Only haemoglobin andplatelet number are stable over thistime. Likewise, stability at refrigeratortemperature is analyzer-dependent (fordetails see Annex). For the reasons giv-en above, the blood smear should beprepared within 1–2 hours of bloodcollection. Extended storage for up to24 hours is not recommended.

Pseudothrombocytopenia

Platelet clumping or agglutination, andplatelets adhering to neutrophils (plateletsatellism (Fig. 23-1)) have sometimesbeen observed with EDTA anticoagu-lated blood, such changes becomingprogressive with the time elapsed aftercollection. This phenomenon elevatesthe white blood cell count while de-

pressing the platelet count. The problemcan be recognized by examination ofthe peripheral blood smear, and alsoby being alerted by the platelet flags or alarms in the instrument (39).Accurate platelet counts on subjectsshowing EDTA-induced platelet satel-lism can be obtained by diluting bloodfrom a finger prick or by collectingblood with citrate as the anticoagulant.

Special considerations for plateletcontent measurements To assess in-vivo activation of plateletsby measurement of constituents in theplatelet α-granules such as plateletfactor IV (PF4), β-thromboglobulin,fibronectin and platelet-derived growthfactor, activation of platelets after bloodcollection should be minimized. Thiscan be accomplished either by prevent-ing the formation of thromboxane-A2 orby maintaining high levels of cyclicAMP within the platelets. An additivemixture used for this purpose consists of0.11 mol/L citric acid, 15 mmol/L theo-phylline, 3.7 mmol/L adenosine and0.198 mmol/L dipyridamole with the fi-nal pH being adjusted to 5.0 (38, 138).PF4 levels in blood collected in this ad-ditive mixture called CTAD is almost 10times less than that obtained in a con-ventional citrate tube (223).

Fig. 23-1 Satellism of plateletson granulocytes (neutrophils)

If a blood collectiontube is drawn to one-half of its nominal volume, the effectiveconcentration of EDTAwould be unsuitablefor preparation of aperipheral blood smearintended for whiteblood cell differentialcount (180).

Blood collection tubesmust have an air spacerepresenting at least20% of the volume ofthe tube to facilitatemixing (170).

Everything from a drop of blood?

56

In clinical chemistry, a number of re-agent- and analyzer-specific problemshave to be considered. Thus manyinterference factors act in a reagent-specific way. In addition there are differences between analyzers andanalytical principles (i.e. ”dry“ and ”wet“chemistry, direct and indirect potentio-metry). In this chapter, a few examplesonly are demonstrated (66, 213, 239).

Special aspects when using so-called “dry chemistry“ (99, 202)When a carrier-bound reagent analyzerfor capillary blood is used, the correctsampling of capillary blood samples isof decisive importance for the reliabilityof the results. The producer of theReflotron® System (Roche Diagnostics,Germany) recommends:

”When a large free-hanging droplethas formed, it should be applied to thesample application field of the test car-rier without directly touching the carrierwith the finger. For an additionalmeasurement it is necessary to prick thefinger at a different site.“Results in a whole blood analyzer maydepend on sample haematocrit, be-

cause the amount of plasma availableto the reactive zone of the test stripvaries at different packed cell volumes.

Dry chemistry methodologies on theone hand offer the possibility of sepa-rating the analyte from many disturbingcomponents in the matrix. On the otherhand the matrix is more diluted in wetchemistry procedures, resulting in loweranalyte and possible interfering con-centrations.

Different results using methods withand without deproteinization?Protein molecules in serum-/plasmaoccupy a defined volume, dependenton the concentration and the size of theprotein. As a result, the concentrationof low molecular solutes (e.g. glucose,electrolytes etc.) in protein-free filtratesare found to be approximately 5%higher than in untreated serum-/plasmasamples without deproteinization. Theinfluence of deproteinization on thedetermination of low molecular weightsubstances which are dissolved inserum-/plasma water, is shown in Fig.24-1 (25). The volume displacementeffect of proteins is especially importantfor the determination of electrolytes us-ing direct potentiometric in comparisonto indirect measuring procedures (88)as well as for determination in depro-teinised whole blood (e.g. glucose).

The volume displacement effect of lipidsA similar displacement effect is observedwith triglycerides in blood. At 5000mg/dL (57 mmol/L) triglyceride con-centration, direct potentiometry givesabout 5% higher (and more true) resultsof electrolytes compared to flame pho-tometry or indirect (after dilution) poten-tiometry (110).

Fig. 24-1Change of concentra-tion of low molecular

weight substancesafter deproteinization

(from (25))

low molecularweight substance

volume ofprotein

57

Special aspects in clinical chemistry

Whole blood versus plasma glucoseWhen comparing glucose in wholeblood with that in plasma, similar, butlarger differences are observed. Sinceblood cells have a higher protein andlipid content and glucose is not equallydistributed between the intracellularand the extracellular space, resultsobtained in plasma are approximately15% higher compared to whole blood,when related to the same volume. TheWHO (3) and the ADA criteria (6) forthe diagnosis of diabetes mellitus aretherefore different for plasma andwhole blood.

Electrolytes (Na+, K+, Cl--, HCO3--)

If during transport and storage of wholeblood the glucose concentration fallsbelow a critical concentration, the cellslose their intracellular potassium andtake up sodium in its place. If CO2escapes from a blood sample, the cellslose bicarbonate and replace it withchloride from the surrounding plasma(chloride shift) (Fig.24-2). This limits thestability of whole blood for the plasma/-serum analysis of electrolytes. Interes-tingly, the increase in plasma potassi-um is higher in refrigerated bloodsamples compared to samples stored atroom temperature (84). This is causedby an inhibition of the cellular Na+,K+-ATPase activity brought about by cold.

Trace elements

Contamination plays a significant rolein trace element analysis. Blood shouldtherefore be obtained in a suitablesampling system that has been declaredtrace element-free by the laboratory.

Lipids

Storage alters triglyceride concentrationdue to the action of endogenous lipases.

The triglyceride concentration in thesample falls while that of free glycerolrises. The extent of this effect varies fromperson to person and does not correlatewith the initial triglyceride concentra-tion. In addition, the composition ofplasma lipids determines their behaviorduring centrifugation. Chylomicrons andtheir remnants tend to float to the toplayer of plasma, whereas other lipopro-teins remain equally distributed. This hasto be considered when primary tubesare used in analyzers (131).

Creatinine

The concentration of non-creatininechromogens increases at room tempera-ture, this effect being more pronouncedin whole blood than in serum/plasma(the higher the temperature, the greaterthe effect). This increase – which variesfrom person to person and is independ-ent of the initial creatinine concentra-tion – occurs when creatinine is deter-mined by the Jaffé method (145).

Recommendation:

In clinical chemical analysis, method-and analyzer-specific prenalytical influ-ences and interferences have to be tak-en into consideration. Results obtainedwith one system are not transferable toothers without experimental proof.

Detailed information may be obtainedfrom reagent and analyzer producers.

Fig. 24-2Electrolyte fluxesbetween blood cellsand plasma/serumduring storage ofwhole blood

plasma/serum

CO2

cellglucose

HCO3 K+

Na+

CI

Special tubes for hormones?

58

Sampling, storage and transportfor analysis by immunochemicalmethods

Sensitive immunochemical methodslend themselves to the measurement oftrace quantities of labile hormones,proteins and other analytes in blood.Because of the wide range of analytesmeasured by immunoassays, this chap-ter will attempt to focus on a few repre-sentative analytes (12).

Posture and timing

Variables such as posture during bloodsampling and diurnal changes have tobe taken into consideration.

Postural variations have a significantimpact on renin, elevation of enzymeactivity being observed when movingfrom the recumbent to the erect position.

Cortisol has a peak value between 4a.m. and 6 a.m (see Fig. 5-2).

Hormones such as growth hormone,lutropin (LH) and follitropin (FSH) are re-leased in bursts, and as such, severalblood specimens taken within closelytimed intervals are needed to establisha median value.

Refrigeration and freezing

Some hormones such as insulin, proin-sulin and C-peptide can be stabilizedby merely placing blood specimen con-tainers on ice immediately after collec-tion. Such specimens should be promptlycentrifuged, preferably in a refrigeratedcentrifuge at 4°C, and the serum keptfrozen until assayed. Of course, the frozenspecimen should be completely thawedprior to assay. It is also important thathaemolysis be avoided, since this will

decrease both insulin and proinsulinvalues.

Processing of blood promptly uponclotting and freezing of serum at –70°Cprovides long-term stability for analytessuch as gastrin, pepsinogen-1, andinsulin.

Collection of blood in anappropriate anticoagulant

Most analytes determined by immuno-chemical methods can be measured inserum and/or heparinized plasma.

Collection of blood in EDTA andpromptly freezing the plasma has beenfound to be adequate for preservinglabile polypeptide hormones such asendorphin, vasoactive intestinal peptide,substance P and pancreatic peptide.

By its inhibitory effect on metallopro-teinases EDTA plasma is also recom-mended for ACTH, parathyroid hor-mone and glucagon.

Collection of blood with proteolyticenzyme inhibitorA proteinase inhibitor called aprotinin(also known by its trade name Trasylol)added to an anticoagulant such as ED-TA or heparin has found application inthe stabilization of labile polypeptidehormones and enzymes (137). Sinceaprotinin inhibits kallikrein, its potencyis expressed in terms of kallikrein in-hibitory units (KIU). The concentrationof aprotinin used for the preservationof labile hormones and enzymesranges from 500 to 2000 KIU/mL.Thus, a mixture of EDTA aprotinin hasbeen used to stabilize glucagon,ACTH, renin and certain gastrointesti-nal hormones such as β-endorphin, se-cretin, neurotensin, gut glucagon, so-

59

Preanalytical factors in immunoassays

matostatin and vasoactive intestinalpeptide (137).

In one study it was demonstrated thatglucagon levels measured by RIA inEDTA plasma were approximately 26%higher than in plasma obtained fromblood collected in a mixture of 1.5 mgEDTA and 2000 KIU of aprotinin per mLof blood (Tab. 25- ). The high gluca-gon level in samples collected withoutaprotinin was due to the fragments ofhormone produced by proteolytic en-zyme degradation being apparentlyrecognized as an intact molecule bythe antibody used in the assay. In addi-tion, some of the radio-labeled hor-mone underwent proteolytic enzymedegradation, thus limiting the amountof radio-label available to competewith the hormone in plasma for bindingsites on the antibody (48).

Tab. 25- Effect of aprotinin on glucagonmeasurements in EDTA plasma

EDTA + Aprotinin EDTA

n 21 21Mean pg/mL 386 518% Difference –25.5

A mixture of lithium heparin andaprotinin has also been used to stabi-lize immunoreactive somatostatin, se-cretin, glucagon, C-peptide andcholecystokinin-pancreozymin.

Collection of blood in an anticoagulant-additive mixture

There are instances where EDTA aloneis not sufficient for stabilizing an ana-lyte. Thus, even when blood is collectedin EDTA and the plasma is separatedpromptly and stored under ideal condi-tions, there is activation of complement.However, when a synthetic protease in-

1

1

hibitor such as nafamostat mesylate isadded to EDTA, the stability of comple-ment components (C3a, C4a and C5a) issignificantly improved. Furthermore,while the activity of complement com-ponents doubles with each freeze-thawcycle when blood is collected with EDTA alone, no such discrepancy isseen in samples collected in EDTA sup-plemented with nafamostat mesylate(227).

Traditional anticoagulants such as EDTAand heparin have been ineffective instabilizing a labile constituent such asparathyroid hormone related protein(PTH-RP). This tumor marker is so unsta-ble that less than 10% of the originalactivity remains after 16 hours of stor-age at room temperature in blood spec-imens collected in either heparin or EDTA (164). The optimum mixture forblood collection is EDTA (1.5mg/mL ofblood), aprotinin (500 KIU/mL), leu-peptin (2.5mg) and pepstatin (2.5mg).With this additive mixture, PTH-RP is sta-ble for up to 1 hour at room tempera-ture and for 24 hours if maintained re-frigerated at 4°C (164).

For the measurement of catecholaminesin plasma, blood should be collected inan anticoagulant mixture such as EGTA(90mg/mL) supplemented with sodiummetabisulfite or glutathione (60mg/mL).Even with this additive mixture, theblood has to be kept cold in an icebath and centrifuged in a refrigeratedcentrifuge. The plasma should then betransferred to a plastic vial, cappedand stored in a freezer at a temperaturebelow –20°C until ready for analysis.Frozen plasma prepared under theabove conditions preserves catechol-amines for at least 3 months (19).

Blood cells can provide important information

60

Separation of cells from peripheralblood for cellular analysis.

For the collection and transport of pa-tient blood samples for cell analysispropylen tubes are required, becausecells are adsorbed on glass and poly-ethylen (178).Separation of a pure population of cellssuch as lymphocytes from peripheralblood are required for tests such ashuman leukocyte antigen (HLA) typingstudies. If the cell preparation is signi-ficantly contaminated with granulocytesand platelets, they may bind some ofthe HLA-antibody thus yielding a falsenegative result.

How to purify lymphocytes

Lymphocytes can be purified using theFICOLL-HYPAQUE centrifugation proce-dure.A typical FICOLL-HYPAQUE mixtureconsists of 10 parts of 33.9% Hypaquewith a density of 1.2 kg/L and 24 partsof 9% aqueous solution of Ficoll. BothHypaque and Ficoll solutions should bemixed at room temperature. The finalFicoll concentration in the mixtureshould be 6.4% and the density of themixture 1.077 kg/L (23).

In the procedure, diluted whole blood islayered over the FICOLL-HYPAQUE mix-ture and centrifuged at room tempera-ture (20 °C) for 40 min. The centrifugalforce attained at the interface is 400 g.Since the FICOLL-HYPAQUE medium isless dense than red blood cells andgranulocytes but more dense than mono-nuclear cells (lymphocytes and mono-cytes) and platelets, the mononuclearcells will remain at the plasma-Ficoll-Hypaque interface. Subsequent washeseliminate platelets from the lymphocytepellet.A recent study described the use ofeither heparin or buffered citrate as ananticoagulant, polyester gel barrierand liquid density gradient medium inan evacuated tube for blood collection.A 20 min centrifugation step at 1,500 gat ambient temperature resulted in iso-lation of peripheral blood mononuclearcells (PBMC) over the gel barrier andseparation from erythrocytes and gra-nulocytes trapped underneath the gel(132, Fig. 26-1).

Effect of anticoagulants on therecovery of granulocytes

Granulocytes can be recovered from FICOLL-HYPAQUE centrifugation by re-

A false negative resultin HLA typing will have

serious consequenceswhen a patient or organ

transplant donor is be-ing typed for HLA com-

patibility.

Fig. 26-1Processing steps with

the cell separationtube

AFTER BLOODCOLLECTIONGently invert 8 times.

1

Acceptablebloodvolume– 8mL– 7mL– 6mL

At room temperatureFor 20 minutesIn a horizontal rotor(swing-out head –appropriate tube adapters)At 1500 g

For best results, centri-fuge within two hoursof blood collection. 2

AFTERCENTRIFUGATION

anticoagulatedwholeblood

gelbarrier

densitygradient

fluid

3

FORTRANSPORTATIONGently invert once.

4

CENTRIFUGE

plasma

lymphocytesand monocytes

gelbarrier

erythrocytesand neutrophils

cellsuspension(plasma andmononuclearcells)density

gradientfluid

61

Special aspects in cellular analysis

moving the fraction above the FICOLL-HYPAQUE layer. 0.4 mL of 4.5% dextranand 1 mL of heparinized plasma arethen added to this fraction. The residualred blood cells are allowed to sedimentat 4°C for 40 min. Dextran promotesred blood cell rouleaux formation andfavors sedimentation. The supernatantis thus enriched with granulocytes.Boyum found that EDTA gave bettergranulocyte yields (average 59%, range43–71%) than heparin (average 49%range 38–61%) (23).

Relative merits of anticoagulantsand nutrients for cell separationand stabilityACD (acid citrate dextrose), heparinand EDTA have all been used for theseparation of mononuclear cells. How-ever, regardless of whether EDTA, ACDor heparin was used as anticoagulant,it has been reported that the lympho-cyte fraction was contaminated withgranulocytes if the blood sample wasover 14 hours old. Red cell contamina-tion is a problem with 2-day-old EDTAblood (161).From other authors it is reported that Fi-coll gradient preparations of mononu-clear leukocytes from EDTA-blood sam-ples are contaminated by neurophileleukocytes and erythrocytes (178).Nutrient dilution media would appearto influence the quality of FICOLL-HY-PAQUE lymphocyte separations. Thus,blood collected in heparin and supple-mented with glutamine and gentamicingave good FICOLL-HYPAQUE separa-tions with blood stored at room temper-ature for up to 3 days (135).

Whole blood lysis for flowcytometry applications

Whole blood lysis techniques for flowcytometric immunophenotyping appli-

cations have, because of their rapidity,supplanted the time-consuming FICOLL-HYPAQUE cell separation procedure(102). Even the lysis techniques have pro-gressed from the original hypotoniclysis techniques to the currently commer-cially available non-hypotonic methods.In a study performed to evaluate theeffects of various anticoagulants on bothlysis methods and the FICOLL-HYPAQUEprocedure, it was demonstrated that ifanalysis is performed on the same day,EDTA, ACD or heparin gave equivalentresults. However, beyond 24 hours thereis a significant decrease in granulocyteviability in EDTA. Furthermore, K3EDTAhas been reported to be associatedwith a loss of function in lymphocytemitogen stimulation assays. Granulo-cyte viability was best for heparin. Lym-phocytes were preserved equally in he-parin and ACD. Although ACD couldbe used as an alternative to heparin,there is a tendency for platelets to ag-gregate in ACD, especially if specimenscannot be analyzed promptly (34).If a flow cytometric test of the leuko-cytes is not performed during six hoursafter sample collection, the cell numbershould be determined immediately af-ter sampling the EDTA blood. That is al-so necessary, if the immunophenotyp-ing is performed with heparin blood(50 IE heparin/mL sample) (178). Onthe other hand, EDTA blood has the ad-vantage that the loss of mature cells ofthe myeloid lineage by adsorption onthe tube wall and platelet aggregationis diminished (178). For the plateletfunction analysis by flow cytometrytechnique the use of citrate blood is re-commended despite some disadvan-tages. EDTA and heparin should beavoided because of their effects onglycoprotein structure and artifactualplatelet activation (185).

How to handle genes

62

Restriction fragment lengthpolymorphism (RFLP)To help understand some of the preana-lytical problems, let us visualize aperson whose DNA is being examinedfor RFLP to detect some genetic defector gene rearrangements. When DNA iscleaved using restriction enzymes, frag-ments of varying sizes result dependingon whether or not the polymorphism iswithin the restriction enzyme cleavagesite (140).

It has been reported that aberrant orunexpected restriction fragments wereobtained on DNA being examined forT and B cell gene rearrangements us-ing blood collected in heparin (88).Such aberrant fragments were notfound upon restriction enzyme digestionof DNA obtained from blood collectedin either EDTA or ACD. Since theseaberrant fragments found using hepa-rinized blood may be confused withgene rearrangements, the choice ofanticoagulant for blood collectionintended for certain molecular biologyprocedures becomes critical (214).

Polymerase chain reaction (PCR)

Heparin at a concentration as low as0.05 U/reaction mixture has been

reported to retard or even completelyinhibit the amplification of DNA duringthe polymerase chain reaction (PCR)(87). However, treatment of heparinizedblood with heparinase to cleave heparinor the separation of leukocytes bycentrifugation followed by at least twowashings with a saline buffer is reportedto overcome the effect of heparin (16).

Actually, other anticoagulants such asEDTA and ACD also inhibit restrictionenzymes. However, standard ethanolDNA precipitation techniques removeboth EDTA and ACD while heparin isnot removed (36).

Heparin should not be used as anti-coagulant in the molecular biologicalanalysis of blood.If heparin is present in sample to beanalysed, it should be eliminated be-fore application of the sensitive rtPCR.This can be done by application of he-parinase or precipitation of mRNA withLiCl (94, 95).

Erythrocytes can likewise inhibit taqDNA polymerase by formation ofhaematin (140).

Red blood cells can be removed byselective lysis with a buffer mixtureconsisting of 155 mmol/L ammoniumchloride, 10 mmol/L potassium bicarbon-ate and 0.1 mmol/L EDTA adjusted topH 4.

Alternatively, the cytoplasmic membraneof all cells can be dissolved with abuffer mixture containing the non-ionicdetergent Triton-X100 leaving behindthe nuclei of white blood cells fromwhich DNA can be extracted. How-ever, this technique will result in the lossof extranuclear DNA and RNA to thesupernatant; hence, mitochondrial DNAwill not be able to be extracted (27).

Fig. 27-1Effect of anti-

coagulants on RFLPs

band 1band 2

band 1

sample collectedin EDTA or ACD

sample collectionin heparin

heparin inhibitoryeffect results inincomplete formation of RFLPs.

extract DNA

incubate with restrictionendonuclease

examine restriction fragmentsby electrophoresis

63

Special aspects in molecular biology

Considerations for the isolation ofDNA and RNA

Classical techniques are based onlysing cells with lysozyme, alkali ordetergents. The removal of proteinsand other contaminants is effected byincubation with protease and/or extrac-tion with phenol or chloroform.

The extract should be concentrated byprecipitation with ethanol in the pre-sence of sodium or ammonium acetate.If necessary, RNA can be removed us-ing DNase-free RNAase.

The advantage of using proteinase K isthat, in addition to releasing DNA fromchromatin, it also destroys nucleaseswhich would otherwise reduce the average molecular weight of DNA(140). However, proteinase K has to beremoved before the isolated DNA canbe subjected to restriction enzyme treat-ment. Also, Taq polymerase can bedegraded by proteases.

Proteinase K can also be inactivated byheating the cell lysate or purified DNAto 95°C for 10 min.

Residual phenol can inhibit Taq poly-merase. Hence, a final extraction withchloroform isoamylalcohol (49:1v/v)should be performed after phenoliza-tion to remove any trace quantities ofphenol remaining in the aqueousphase.

Salts used to subsequently precipitateDNA should be removed by washingthe pelleted DNA with 80% ethanol.

The type of detergent used for cell lysismay influence DNA amplification byPCR.

Generally, non-ionic detergents such asTween 20 and Triton X-100 do notinhibit Taq polymerase in concentra-tions of less than 5% (v/v).

However, ionic detergents such assodium dodecylsulfate (SDS) which aregenerally used in concentrations up toas high as 2.0% (w/v) can be inhibito-ry to Taq polymerase, since a concen-tration greater than 0.01% (w/v) hasbeen found to be inhibitory.

Other ionic detergents such as sarkosyland sodium desoxycholate have beenshown to inhibit Taq polymerase atconcentrations greater than 0.02%(w/v) and 0.06% (w/v) respectively.Hence, it is important that ionic de-tergents be efficiently removed byphenol/chloroform extraction and byethanol precipitation and subsequentwashing of the DNA pellet.

Even with non-ionic detergents such asnonidet P40 (NP40), where 1% (v/v)has no effect on reverse transcriptaseenzyme, 0.1% (v/v) can inhibit Taqpolymerase.

Hence, it is important to perform pre-liminary experiments to establish theeffective concentrations of detergentsand other known inhibitory reagentsthat may affect DNA amplification byPCR.

Chaotropic agents such as guanidi-nium isothiocyanate have been fre-quently used for the extraction of DNAor RNA.

The advantage of using 5 mol/Lguanidinium isothiocyanate for RNAisolation is that it is able not only toremove proteins from RNA but also todenature ribonucleases which wouldotherwise degrade RNA (140).

How to handle genes

64

Stabilizing RNA during transport,storage and sample preparation

A number of factors influence RNAstability. This rapid decrease in reversetransscriptase PCR sensitivity has beenshown to be due to RNAses present inthe sample, limiting the time of process-

ing of native samples to 2 hours (140).By using 5 mol/L guanidinium isothio-cyanate, the sample is stabilized by de-naturation of RNAses for approximate-ly one week at room temperature.

In terms of isolation of undegradedRNA, one must eliminate ribonuclease(RNase) contamination. These enzymesare so stable over a wide range of pHand resistant even to boiling thatglassware, reagents and even the in-vestigator‘s fingers are a source ofpotential contamination. Glasswareshould be treated with a 1% solution ofdiethylpyrocarbonate (DEPC) which isknown to inhibit RNases. Residual DEPC, however, should be thoroughlyremoved by autoclaving the glasswarein order to convert DEPC to carbondioxide and water and subsequentheat-treatment of the glassware at250°C for 4 hours.

Even sterile disposable plastic ware isnot always free of RNAse! In case ofcontamination the materials like pipettetips and tubes should be autoclaved inhot air before use.

Contamination control

DNA from exogenous sources such asa person’s hair or skin, door knobs,laboratory bench, dust, reagents, ther-mocycler and pipette tips are commonsources of contamination.Ideally, a laminar air flow bench withfiltered air provides a clean dust-freeenvironment.

Sample preparation should be carriedout in a separate room or area.

Recently, Neumaier et al. have publishedrecommendations on quality assessmentof molecular biology diagnostics (160)desribing preanalytical aspects:

Fig. 27-2Use of anticoagulants

and stabilizers insampling of blood for

gene analysis

a

b

c

RFLPandPCR!in

HEPARIN

RFLPandPCR

inEDTACitrate

orACD

!

instabilizing

RFLPandRNA

in5 mol/L !guani-diniumisothio-cyanate

65

Special aspects in molecular biology

Specimens: PCR analysis can be ap-plied to EDTA and citrate blood, driedblood (filter-paper cards), bone, mar-row, buffy coat, sputum, mouthwash,bronchial lavage, cerebrospinal fluid,urine, stool, biopsy material, cell cul-tures, fixed or embedded tissue etc.).Depending on the test material, a pre-treatment of the sample may be neces-sary prior to stabilization, e.g., lique-faction of sputum.

Sampling: sampling is best done in closed disposable sampling systems asare customary with other clinical testmaterial. New disposable plasticwareis considered to be free of nucleases.When using nonclosed sampling sys-tems, at least disposable gloves are tobe worn.

Sample stabilization: stabilization of testmaterial is essential as nucleic acidsdegrade rapidly. This is especially im-portant where RNA is to be analyzed.The fast inactivation of DNases andRNases is achieved reliably by chaotro-pic substances (here especially guani-dinium-isothiocyanate, GITC). Organicsolvents, e.g., phenol, may be added inparallel. Extraction systems with thoseadditives are commercially available,e.g., RNazol, Trizol. The limited stabilityof reducing substances (here: β-mercap-toethanol) and their effect on sample sta-bility is to be considered. The user hasto be aware that batches of ready-to-use extraction solutions have limitedshelf life due to instability of single com-ponents, e.g., β-mercaptoethanol. En-riched cells or native specimens of sin-gle components are lyzed by additionof GITC. The final concentration of GITCin the stabilized sample should not falllower than 4 mol/L. Material stabilizedin this manner needs not to be cooled.At temperatures lower than room tem-perature, GITC may crystallize. EDTA

blood for the extraction of DNA fromleukocytes requires no special stabiliza-tion.

Sample dispatch: samples stabilized ap-propriately may be dispatched by postat room temperature. This applies alsoto EDTA whole blood for DNA prepara-tion and GITC stabilized samples forRNA recovery. Cooling is not neces-sary, but depending on the applicationthe prolonged storage at room temper-ature will result in a critical loss in sen-sitivity. Samples are to be dispatched inbreakproof containers. Non-stabilizedsamples must be shockfrozen and thanbe dispatched in dry ice. The coolingchain must not be interrupted.

Sample storage: specimen for DNAanalysis are to be stored in 10 mM TRIS,1 mM EDTA, pH 7.5–8.0 at 4°C. Speci-mens for RNA analysis should be keptin buffered solution preferably at–20°C. GITC stabilized RNA samplesmay be stored for approx. seven daysat room temperature.

In conclusion, understanding the prean-alytical pitfalls in molecular biology ap-plications and minimizing or elimina-ting them is a prerequisite to thesuccessful utilization of such techniques(95, 119, 143, 160, 190).

Fig. 27-3Microchip PCR asdiagnostic tool

AAAACGACAC

AAAACGACAC

AAAACGACAC

AAAACGACAC

CGGTCGCCGC

CGGTCGCCGC

CGGTCGCCGC

Nucleotides

}n10 n10 n10 n10 n10 n10 n10

T = ThymidineG = GuanineC = CytosineA = Adenine

Carrier

Spacer

When gases evaporate

66

Preanalytical variables in blood gas,acid-base and electrolyte measurementhave recently been addressed in IFCCrecommendations (30).

Anticoagulant

Heparin is the recommended anti-coagulant for blood gas and electrolytedeterminations in whole blood. Drysodium heparinate may increase sodium,decrease pH, bicarbonate and baseexcess. In addition, ionized calcium isdecreased if binding sites on heparinare not saturated. The recommendedfinal heparin concentration in blood dif-fers for glass and plastic tubes (195).

Specimen collectionThere is a difference in blood gasesand acid-base status between arterialand venous blood due to metabolism inthe respective limb or tissue. In general,oxygen is consumed, CO2 is increased

and pH is decreased due to respiratoryand metabolic components along thearteriovenous blood flow. Arterial andcapillary sampling sites should thereforebe preferred to obtain systemic acid-base and blood gas results. If in-dwelling catheters or cannulae areused for sampling, it has to be ensuredthat fluid or flush solutions are removedcompletely from the system by with-drawing a volume equal to three timesthe volume of the catheter system priorto blood collection (151). Blood shouldbe collected anaerobically to preventgas exchange with the surrounding air.Venous occlusion by tourniquet shouldbe restricted to a maximum of 2 minutes.Formation of bubbles should be pre-vented. The sites recommended forsampling blood are those described inthe chapters on arterial and capillarysampling (see p. 20–23).

Recommendation on capillarysampling

When sampling from skin, the firstdrop should be removed and bloodallowed to flow into the he-parinized capillary without anysqueezing. The tip of the capillaryshould be placed deeply into thedrop and filled completely withoutany pressure while holding in ahorizontal or slightly downwardposition. The capillary in the dropshould be closed off immediatelywith a plastic cap followed by in-sertion of a metal mixing bar(”flea“). The opposite end of thecapillary should then be sealed.Blood and heparin are mixed bymoving the bar with the aid of amagnet from end to end five times.

Recommendations on the useof heparin in blood gas andelectrolyte analysis

Heparin solution may be added ata final concentration of 8–12 IU/mLin glass and 4–6 IU/mL in plastictubes. The final concentrationranges for buffered heparin solu-tions should be, in mmol/L:

Sodium: 120–150Potassium: 3.5–4.5Ionized calcium: 1.2–1.4Chloride: 100–130.

The pH of the heparin solutionshould be between 6 and 8 (30).

67

Special aspects for blood gases and ionized calcium

Fig. 28-1Alteration of pO2 inwhole blood (pO2 =85 mm Hg =̂ 11.3 kPa)stored in a plastic orglass syringe for 45min at room tempera-ture (mean and SD of15 measurements ineach type of syringe)(133)

4.8

3.2

1.6

00.4

–0.8

36

30

24

18

12

6

-6

∆pO2(kPa) (mm Hg)

10 20 30 400 t (min)

plastic syringe

glass syringe

50

Storage and transport

Specimens for blood gas and elec-trolyte measurements may be affectedduring storage by the following pro-cesses (133):

● Metabolism of blood cells: glycolysis,mainly in red cells, causes formation oflactic acid and shifts in pH, bicarbo-nate and base excess towards therange of metabolic acidosis. Oxygenconsumption in leukocytes and plateletsdecreases pO2 and increases pCO2.The fall in pO2 is accelerated if theoriginal sample pO2 is elevated. Themetabolic processes can be reducedby cooling the sample. Cooling isnecessary if the sample cannot be ana-lyzed within 15 min of sampling.

● Ion release from blood cells: pro-longed storage, vibration during sampletransport and severe thrombocytosisare factors which may contribute to anincrease in potassium and a decreasein ionized calcium in the plasma.During the first hour of storage in icewater, the average increase in plasmapotassium concentration is 0.1 mmol/L(133).

Ionised calcium can be stabilised up to8 hours after separating plasma bycentrifugation (96).

● Gas exchange: As mentioned above,plastic materials are not absolutely gas-tight. It has been observed that refrig-erated plastic tubes leak more gasesthan those kept at room temperature. Acompromise has therefore been sug-

gested that storage in ice water shouldnot exceed 30 min when stored inplastic. Glass capillaries and glasssyringes remain gas-tight for severalhours (133) (Fig. 28-1).

Sample preparation

Upon arrival of samples for blood gasand acid-base measurement, carefulremixing of the samples prior to analysisis necessary.

Specimens in glass capillaries shouldbe remixed by moving a metal wirefrom end to end for 5 to 10 sec. Glassor plastic syringes should be inverted10 times and then rolled horizontallyfor 10 sec (30). No bubbles or deadspace should be formed during mixing.Mixing the sample is of special impor-tance if haemoglobin is measured si-multaneously (134).

The right time for drugs…

68

Which sample for TDM?(49, 124, 136)

The most commonly sampled body flu-ids are blood, plasma and serumbecause a good correlation betweendrug concentration and the therapeuticeffect can usually be found. In addition,TDM in plasma/serum is useful inpreventing toxic side effects of the drugby overdosage or decreased elimina-tion (metabolism).

In special cases, urine is also useful asa sample material; saliva and CSF areanalyzed less frequently, although theymay better represent the free drugconcentration in some cases.

Sampling – a matter of timingThe optimal sampling time varies withthe drug and the dose schedule used(see Tab. 29- and Fig. 29-2).

When making an estimate of ameaningful interval to select betweentwo determinations, it should beremembered that approximately fivehalf-lives are necessary for equilibriumto be reached in the body betweenintake and elimination, i.e. beforeadapting the dosage one should waituntil at least this interval has elapsedbefore the next measurement of thedrug concentration. Maxima are givenin Tab. 29- , minimum concentrationsshould be obtained shortly before thenext dose is applied (Tab. 29- ).

Tab. 29- Rules for blood sampling in TDM

Time at which blood sample should be taken

Long-term therapy Basically always in the steady-state(after approx. 5 half-lives)

Intravenous One must wait until the distributionadministration phase is completed (approx. 1– 2

hours after completion of the infusion)Exception:Digoxin 6 – 8 hoursDigitoxin 6 – 8 hours

For further details, see the NCCLS-document on therapeutic drug monitor-ing (148).

Which specific aspects of samplingare important for TDM?Blood should not be taken from the arminto which drugs or transfusion fluidsare being infused.

Heparin, EDTA, or potassium oxalatecan be used as anticoagulants. How-ever, heparin leads to a change in

1

2

2

2

Fig. 29-1The elimination of

most pharmaceuticalstakes place in accor-

dance with a first-orderreaction, i.e. a certainpercentage of the totalquantity in the body iseliminated per unit of

time irrespective of theadministered dose andthe concentration of thepharmaceutical agent

in the sample (167)

concentration in blood

0 4 8 12 16 20 24 28

8

4

0.51

2

time of day

Fig. 29-2Concentration of theo-phylline in plasma af-ter oral administrationof pills either as imme-diate-release formula-tion (red points) or assustained-release for-

mulation (blue points).The immediate-release

formulation was ad-ministered 3 times aday (at 6.00, 15.00

and 22.00 h), the sus-tained-release formula-

tion 2 times a day at8.00 and at 20.00 h

(53)

15

10

5

0800 1200 1600 2000 2400

time of day

concentration in plasma (mg/L)

69

Special aspects in therapeutic drug monitoring (TDM)

protein binding in some drugs (236).EDTA is believed to be the optimal anti-coagulant and should be the agent ofchoice for measuring levels of tricyclicantidepressants since, by chelatingdivalent cations, EDTA might contributeto the stability of these drugs by protect-ing them from oxidation (147).

Haemolyzed blood samples are pre-ferred to serum or plasma for cy-closporine determination, because nu-merous factors (temperature, haema-tocrit, concentration of lipoproteins) af-fect the blood/plasma relationship of ciclosporine (238). EDTA is also re-commended as an anticoagulant.

Some immunoassays for drugs can bedisturbed by unspecific cross-reactionsof endogenous interference factors,e.g. digoxin-like immunoreactive sub-stances (steroids, lipids) may interferewith digoxin (falsely elevated results).

Sampling of urine should be carriedout over at least 7–10 biological half-lives (see Annex and (52)), which coversmore than 99% of the excretion phase.

Sampling times for saliva should besimilar to those for blood. The mouthshould be thoroughly cleansed withwater before sampling. The saliva mustbe completely free of food particlesand any drug retained in the mouthfollowing oral administration.

What about storage and transport?

Disposable containers should be usedto collect the specimens in order toreduce the possibility of contamination.Some plasticizers released from plasticsyringes or from the rubber closures ofglass containers may affect assayresults (191).

To ensure their integrity, the samplesshould be sent to the laboratory withoutdelay. To avoid haemolysis and decom-position, only plasma, serum, urine orCSF should be sent. Nitrazepam, chlor-azepam and cocaine, for example,have been found to decompose duringstorage of blood samples at 4°C.

For ciclosporine A (CsA) which parti-tions into the red blood cell rapidlywhen blood cools from body tempera-ture (37°C) to room temperature (20°C)after collection, whole blood is thespecimen of choice. The preferred anti-coagulant for monitoring CsA is EDTA(192).

When plasma is separated at roomtemperature, the plasma to blood ratiofor CsG is 0.8, whereas for CsA it is0.6 (147).

How stable are samples atdifferent temperatures?Stability of samples for TDM is summa-rized in the “List of Analytes“ (seeannex). Strict observation of the kit pro-ducer‘s instructions is recommended.

The freezing of blood for the deter-mination of ciclosporins should bestrictly avoided. In addition, samplesfor fluorouracil need to be collected onice (unstable at room temperature).Frozen samples should preferably bethawed at room temperature. Too rapidthawing by warming the sample maycause overheating and decomposition.Thorough mixing is very important inorder to secure homogeneity prior toanalysis.

The right time for drugs…

70

Tab. 29- 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 collectionconcentration

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-releasepreparations

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 metaboliteProcainamide/ 1– 4 h (oral) 15 – 25 h 3 – 5 h approx. 15 % Maximum immediately after the lastN-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 administrationTobramycin* administration; 2.5 –15 h 0.5 – 3.0 h Trough level just before subsequent Netilmicin 30 min after i.v. > 30 y of age 2–3 h injectionAmikacin administration 7.5 – 75 h 1.5 – 15 hVancomycin 30 min 20 – 30 h 4 –10 h 30 –55 % Peak 30 min after infusion of 1 hStreptomycin 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 unimportantEthosuximide 2 – 4 h 7 –14 d 10 – 60 h 0 %Phenobarbital 6 –18 h 10 – 25 d 50 –120 h 50 % Actual sampling time unimportantPhenytoin 15 – 30 h 8 – 50 d 25 – 200 h 92 % During the dosage interval; actual

Sustained-release preparations 3 – 9 h sampling time unimportantPrimidone 0.5 –7 h 2 – 4 d 6 – 8 h 35 %

Phenobarbital 10 – 25 d 48 –120 hValproic acid 2 – 8 h 2 – 3 d 8 –15 h 90 %

2

▲▲

≤ 10 %

71

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 collectionconcentration

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 ingestionDigoxin 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 takenNortriptiline 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)

Bacteria and viruses

72

The diagnostic value of a microbiologi-cal test is influenced by a number ofpre-analytical factors (129, 184). At-tention must be given to the following:

● Unambiguous diagnostic question● Precise details of the collection site● Correct method and time of collec-

tion● Suitable transport systems● Shortest possible transport times● Correct storage of the specimens

before processing

Various containers for the collectionand transport of specimens for microbi-ological testing and the requirementsfor such containers are summarised inFig. 30-1 and Tab. 30- .

Even the most sophisticated transportsystem can never be a substitute forshort transport times and immediatespecimen processing.

Tab. 30- : Requirements for specimen containers for the transportation ofmicrobiological specimens

1. Sterile2. Non-corrosive3. Sufficiently large4. Secure closure5. Unbreakable6. Transport media

without growth enhancers orwith culture media for certain organisms

BacteriaWhen collecting specimens for bacterio-logical testing particular care is to betaken to avoid contamination. Beforeaspiration the skin must always bemeticulously disinfected. Purulent le-sions should be aspirated through theskin, if possible, as this is easier to dis-infect than the mucous membranes. Liq-uid material is more suitable for micro-

1

1

biological testing than swab specimens.Aspirates are delivered to the laborato-ry in the syringe after the needle hasbeen removed and the syringe securelycapped. In the case of open wounds,the superficial secretions should bewiped off to remove interfering secon-dary organisms and a swab specimenthen collected from the margins of thewound. Swab specimens must be pro-tected from drying out during transport.This can be done by placing the swabeither in a liquid broth or in a transportmedium. In the case of low bacterialcounts the volume of the specimen shouldbe as large as possible. Specimens forblood cultures if possible should be col-lected while the fever is rising. If infec-tive endocarditis is suspected up to tenblood cultures must be collected.

Short transport times and variable trans-port or storage temperatures are impor-tant for several reasons. Refrigerationand pH changes of the specimen or ex-posure to oxygen reduce the survivaltimes of a number of organisms such asmeningococci, gonococci, Haemophilus,pneumococci, Bordetella, Salmonella,Shigella, cholera vibrios, Helicobacterpylori and anaerobic organisms. Whilethe viability of these environmentallysensitive organism can decrease rapid-ly during the transport, others multiply if the transport times are too long. Thismakes the quantitative evaluation of aculture difficult (urine). However, thesought organism can also become over-grown with other organisms. All speci-mens for bacteriological testing shouldtherefore be delivered to the laboratorywithin twohours after collection. The trans-port and storage requirements for bac-teriological testing are summarised inTab. 30- . If these conditions cannotbe met, inoculation of a culture bottle isrecommended or, for urine samples forexample, the use of dipslides.

2

73

Special aspects in microbiology

Fungi (130)

To obtain skin specimens for mycologi-cal testing, scrapings from the activeareas of the lesions, i.e. the edges, arecollected with a scalpel after thorough-ly disinfecting the skin area. Skin, hair(collected from the edges with epilationpipettes or excision off in the case ofdeposits on the hair) and nail clippingsshould be sent to the laboratory dry insterile containers. Scrapings from theunderside of the nail are used for cul-ture. For detection of yeasts in urine, arandom urine specimen should be sent

to the laboratory promptly in a sterilecontainer. The same applies to the de-tection of yeasts or moulds in sputumspecimens with morning sputum speci-mens being preferred. Tissue speci-mens for mycological, as for bacterio-logical, testing should be placed inisotonic saline and sent to the laborato-ry as rapidly as possible. For mycologi-cal testing of the vagina, the upper res-piratory tract or the stool, submission oftwo swab specimens in sterile contain-ers is recommended. Specimen trans-port at room temperature is usually un-critical for mycological cultures if the

Tab. 30- : 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 notCerebrospinal fluid specimen in (capped) syringe incubate, protect from coolingPleural, pericardial, under anaerobic conditions.peritoneal, synovial fluid Delayed transport: use Paranasal sinus secretions transport medium

Bronchoalveolar lavage (BAL) fluid Prompt transport (2–3 h) CoolSputum, other secretionsStool

Urine Dipslide Room temperature or 37 °C

Swab specimens fromEyesEarsMouthThroat Swab in transport medium Room temperature,Nose (> 4 h transport time) do not incubateUrethraCervixRectumWounds

Biopsy material Prompt transport in sterile Coolisotonic saline

2

Bacteria and viruses

74

transport times are short. In the case oflong transport distances refrigeration ofthe specimens (not necessary for swabspecimens) is recommended in order toprevent overgrowth of the slowly grow-ing fungi with bacteria. If a phycomy-cete infection is suspected (e.g. Mucor)rapid specimen transport without refrig-eration is necessary.

Parasites (92)

Specimens examined for the diagnosisof parasitic infections are blood (plas-modia, trypanosomes, leishmaniae, mi-crofilariae, Loa loa), stool (Giardia, cil-

iates, helminths, cestodes), tissue speci-mens of the affected organs (Trichinellaspiralis larvae, Echinococcus) or theparasites themselves (arthropods: ticks,mites, insects). In the case of stool spec-imens it should be noted that the vege-tative stages are unstable. These stagescan only be detected by examinationof fresh, body-warm stool. Cysts arestable. MIF (merthiolate-iodine-formalin)and SAF solution (sodium acetate – for-malin) have become routine for para-site concentration and preservation instool specimens. For most specimenstransport is uncritical and special trans-port conditions need not be observed.

Tab. 30- : 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, proglottidescomponents 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 intestinalnematodes, in some cases Echinococcushooklets

Skin Skin snip in isoton. NaCl (I) Onchocerca (microfilariae)Sterile skin biopsy Leishmania

Recovery of eggs or Cellulose tape technique Pinwormsadults from perianal skin

3

75

Special aspects in microbiology

Refrigeration of the specimens is rarelyever necessary. Arthropods are sent tothe laboratory in 70% alcohol. Tab.30- gives a brief overview of preana-lytical factors important for parasitolog-ical testing.

A fundamental prerequisite for a para-sitological examination is a travel histo-ry (place, time and duration of visit)and information on onset of symptoms,treatment and whether or not the pa-tient is immunosuppressed. A requeststating “test for parasites” is inade-quate.

Viruses

The time of specimen collection for virusisolation and identification is critical.Usually the material should be collectedimmediately after the onset of symptoms(if possible within the first three days).As a general rule, specimens should bedelivered to the laboratory rapidly at4°C in an insulated container. Virusesusually remain stable for 2 to 3 daysunder these conditions (130). Swabspecimens (nose, throat, eyes), pharyn-geal washings, vesicular fluid from skinlesions, stool, urine and CSF are usedfor analysis.

Specimens for microbiological studieswhich do not meet certain standards(see Tab. 30- ) should either be reject-ed or only analysed after consultationwith the requester.

4

3

Tab. 30- : Cases in which the specimen shouldonly be processed after consultationwith the requester (129)

1. No label/identification2. Abnormally long transport time3. Unsuitable or leaking containers4. Unsuitable specimen5. Submission of the same specimen material with the

same question or request within 24 hours(except blood samples)

4

Fig. 30-1 Containers for trans-port of specimens formicrobiological studies

Specimen tube for detection oftubercle bacilli

CSF/Urine/AspiratesSputum

Swab tube Swab set with trans-port culture medium

Blood culturebottle

Tracheal aspiration set Stool

Can turbid samples be used?

76

The lipemic samplePlasma and serum samples are some-times turbid to varying degrees due toan increased lipoprotein content (Fig.31-1). In nearly all cases, turbidity iscaused by an increased triglycerideconcentration. The turbidity may beslight, often called opaque, translucent,turbid or milky. The degree of turbiditydepends not only on the amount oftriglycerides but also more markedly onthe presence of macromolecular spe-cies of lipoproteins. Turbid samples aretherefore called lipemic.

The diagnostic importance of turbidity

Because normal samples do not exhibitany turbidity except after a fatty meal,the turbidity of a sample is always ofclinical relevance and should be as-sessed, documented and reported bythe laboratory (73 and Annex). It mayindicate hypertriglyceridemia due to anincrease in chylomicrons, very low den-sity lipoproteins (VLDL) or both. As de-scribed in textbooks, these forms canbe differentiated by observing the float-ing of lipoproteins during centrifuga-tion and storage (31, 66, 213). A dis-tinct creamy layer floating over a cleanlayer after centrifugation on storageover at least 12 hours in the refrige-rator indicates the presence of chylomi-

crons. In contrast, a more homogene-ous turbidity is in most cases caused bythe presence of increased concentra-tions of VLDL.

The concentration of triglycerides lea-ding to turbidity depends on the com-position of lipoproteins. Chylomicrons,due to their size, deflect light at de-tectable rates even at triglyceride con-centrations below 300mg/dL (3.4mmol/L). However, the intermediateand low density lipoproteins can be in-visible even at triglyceride concentrationof 800mg/dL, or higher. Varying de-grees of turbidity are observed withVLDL, depending on their size and com-position (10, 60).

Relevance of turbidity as an interference factorWhereas the degree of hyperlipidemiais of diagnostic relevance, the inter-ference of lipoproteins with the deter-mination of lipids and other blood con-stituents should be regarded as distur-bing interference factors which shouldbe avoided as far as possible.

Mechanisms of interference

The following mechanisms have beenfound to cause either falsely low orhigh laboratory results:● Inhomogeneity: Triglyceride-rich lipo-proteins cause them to float during cen-trifugation and storage of serum/plasmasamples. When analyzed after suchtreatment (centrifugation) without carefulmixing, triglycerides and other constitu-ents may be inhomogeneously distri-buted in the sample. This may cause adisproportionately high concentrationof lipids in the upper layer and causeinterference in other methods like totalprotein. On the other hand, lipids maydisplace water in the upper phase of a

Fig. 31-1Plasma samples withdifferent degrees of

turbidity


77

Effects of lipemia

sample thereby leading to a lower ap-parent concentration of water-solublecomponents like electrolytes and meta-bolites.

● Water displacement is also respon-sible for the higher concentration ofsodium and potassium observed indirect ion-sensitive electrode measure-ment compared to flame photometry(110). In exceptional cases, lipids candisplace up to 10% of the water contentof a serum/plasma sample.

● Interference by turbidity: Photometricprocedures are sensitive to turbidity atnearly all wavelengths (65). This leadsto various degrees of absorption (Fig.31-2).

● Interference by physicochemical me-chanisms: Lipoproteins in the samplemay incorporate lipophilic constituents,thereby decreasing their accessibility toantibodies. Likewise, electrophoretic andchromatographic procedures may bedisturbed by lipoproteins.

“Diagnosing” and “treatment” ofinterference due to turbidity

Relevant turbidity can easily be detect-ed with the naked eye. Alternatively,the turbidity of each sample can bemeasured by automatic analyzers usinga specific wavelength (660–700 nm)(65). The degree of interference of each method can be quantified byadding different amounts of samplefrom a hyperlipemic patient to a clearsample subsequent to analyzing theconcentration of both samples sepa-rately. When a test is known to be dis-turbed by any of the mechanisms men-tioned, triglycerides may either beremoved from the sample by ultracen-trifugation (14) or precipitation (114)

and the analysis repeated with theclarified sample.

Care has to be taken regarding theclarification procedure itself as a pos-sible cause of interference.

In some cases a change in methodolo-gy may be helpful in eliminating inter-ference due to lipids. Thus, a secondwavelength may compensate for turbidity.Alternatively, a blank sample may berun without the relevant reactant butunder otherwise identical conditions. Ineach case the degree and type of turbi-dity should be documented and reportedand an aliquot of the untreated samplestored for later testing for verificationpurposes.

Procedures for treating lipemic samplesshould be documented in the quality as-surance manuals of each laboratory.Producers of test kits should indicatethat interference by lipemic sampleshas been tested for and provide respec-tive information in the product leaflet(73 and Annex).

triglycerides added mg/dL

2.0

1.5

1.0

0.5

60 120 240 480 960

uric acid

protein, glucosechloridesodiumcreatine kinasecreatinine

relativeconcentration

0

Fig. 31-2Method – dependentinferference of variousanalyte determinatinsby increasedtriglycerides (65)

A difficult case

78

ues are low, resulting in high MCH andMCHC values. The leukocyte andthrombocyte counts are falsely eleva-ted, because the agglutinates depen-ding on their size are either counted inthe leukocyte or thrombocyte channel.The blood smear shows agglutinationof erythrocytes. Both the determinationof blood group and crossmatching pro-cedures can be affected by cold agglu-tination in two ways. First, panaggluti-nation introduced by antibodies caninfluence the correct assignment ofblood group antigens and the cross-matching procedure. Second, the coldagglutination antibodies may mask other types of antibodies which mayaffect analytical procedures in a differ-ent manner.

Cryoglobulins

Cryoglobulins crystallize in sampleskept at room temperature. The resultingparticles are of varying shape and maymimic leukocytes, resulting in a falselyelevated leukocyte count (Fig. 32-2).Moreover, high concentrations of cryo-globulins can affect erythrocyte count,haemoglobin determination (floccula-tion phenomenon) and platelet count(pseudothrombocytosis) (Fig. 32-2). Theblood smear shows flocculated darkblue crystals without elevated leukocytenumbers. The phenomenon of pseudo-leukocytosis depends on the length ofcontact, temperature, concentration ofcryoglobulins and the interaction ofcryoglobulins with other plasma pro-teins (1). This is valid for all cell numberdeterminations by particle counting.

EDTA-dependent antibodies

Antibody-induced falsely decreasedplatelet counts (without haemorrhagicdiathesis) can be due to cold agglu-tinins or antibodies active in presence

Cold agglutininsAntibodies as interference factors inclinical chemistry are often neglectedsince detection of this factor is difficultunder day to day routine conditions.Analytical procedures in clinical chem-

istry, haematology and immunohaema-tology can be affected by antibodies(107). Antibodies may affect the cellcount of erythrocytes, leukocytes andplatelets (234). High titers of cold ag-glutinins directed against erythrocyteslead to agglutination. Such agglutina-tion alters the electronic cell count inthe following way: erythrocyte count islow at normal haemoglobin concen-trations; MCV is grossly enhanced(Fig. 32-1); calculated haematocrit val-

Fig. 32-1“MCV”-determination

of blood in coldagglutinin disease at

different temperatures(15)

Fig. 32-2Distribution of cryo-globulin particles at

different temperaturesand the corresponding

increase in leukocytecount at different

storage times at roomtemperature (1)

100

75

50

25

30 60 90 120 150 180 210cell volume (fL)

20°C31°C

36°C37°C

frequ

ency

(%)

particles G/L

particle diameter (µm)

37°C

21

3,7

5,8

9,2

14,6

3,7

5,8

9,2

14,6

876543

4°C 25°C

2,9

4,6

7,3

11,6

18,5

5040302010 1 2 3 4 5 6 time (h)

leukocytes G/L

79

Pitfalls with endogenous antibodies

resis. Both types of macro CK may affe-ct accurate quantification of CK-MB bymeans of CK-M-inhibiting antibodies re-sulting in falsely elevated CK-MB activi-ties. Another example is macroamylasewhich is characterized by enhancedactivity in serum while urinary amylaseexcretion is unchanged (209).

Autoantibodies

Immunoassays can be affected by au-toantibodies or heterophilic antibodies(21). Well-described examples are au-toantibodies directed against triiodo-thyronine and thyroxine. Thyroid hor-mone concentrations are apparentlyenhanced since the tracer is bound notonly to the receptor antibody added tothe sample but also to the autoanti-body. Antiphospholipid antibodies inplasma results in increased APTT valuesbecause the antibody binds phospho-lipids used as reagent in the assay.

Heterophilic antibodies

Heterophilic antibodies are detected insome human serum samples, the me-chanism underlying generation of theseantibodies being unknown.In some cases, interference by hetero-philic antibodies can be of diagnosticsignificance. If antibodies have anti-mouse specificity and assays employ-ing immunoantibodies from mice areused (murine monoclonal antibodies),interference of these assays is possible.There are several reports in the literaturedescribing wrong therapeutic measuresas a consequence of such antibodyinduced analytical errors (21). In thiscase monoclonal antibodies caused theinterference. However, antibodies havebeen described, which are unspecificand interfere as well. Only the latter are classified as heterophilic antibodiesin the narrow sense (116).

of EDTA. In both cases, agglutinationtakes some time. Thus, a prolongeddelay between obtaining the sampleand platelet counting results in a morepronounced pseudothrombocytopenia.Platelets of patients with thrombasthe-nia, which lack the membrane glyco-proteins IIb and IIIA, do not react withEDTA-dependent antibodies. This obser-vation suggests that these glycoproteinsare actively involved in binding theantibody. Depending on their shapeand volume, thrombocyte aggregatesmay be counted as leukocytes. In addi-tion to pseudothrombocytopenia elevat-ed cell counts for leukocytes may beobserved. Detection of particles of thesize of lymphocytes in the white cellhistogram is evidence of a spuriouscount of leukocytes. Staining of peri-pheral blood allows detection of aggre-gates of platelets. Other causes offalsely decreased thrombocyte countsare platelets adhering to leukocytes(platelet satellism), giant platelets, oranalysis of partly coagulated bloodsamples caused by a wrong samplingtechnique.

“Macroenzymes”

The possibility of complexes with immuno-globulins (macroenzymes) has beendemonstrated for all diagnostically rele-vant enzymes. A consequence of suchphenomena is an increased biologicalhalf-life of such enzymes. The increasedhalf-life may in turn result in enhancedenzyme activity which can provokefurther diagnostic measures. The pheno-menon of macroenzymes is primarilyobserved in elderly patients with chronicdiseases. Well-described examples aremacro creatine kinase (CK) type I andtype II. Macro CK type I is an immu-noglobulin CK-BB complex. Type II re-presents polymers of mitochondrial CK,which can be detected by electropho-

The serum sample looks reddish

80

Haemolysis is not always accompaniedby the release of haemoglobin (forinstance, if blood is stored at low, butnon-freezing temperature). Interferentsmay also arise from both platelet lysisand granulocyte lysis.

Mechanisms of interference (73 and Annex)The effects of haemolysis may be classified according to the mechanisms involved:

● Increase of intracellular constituentsin the extra-cellular fluid. The efflux ofintracellular constituents may occur in-vivo, during sampling and at all stagesof the preanalytical phase. According-ly, haemolysis may be a diagnosticallyrelevant observation, defined as an in-vitro influence factor when occurringduring sampling or other steps of thepreanalytical phase as it leads to alter-ation of the sample composition. Fig.33-2 shows the effect of increasinghaemolysis on various serum analytes.

● Optical interference may be due tothe colour of the haemoglobin, whichmay change during sample storagedue to haemoglobin formation. The di-rection and degree of interferencediffers not only with the wavelength(s)but also with the type of blank andreagent used. Recently therapeuticallyapplied artificial oxygen carriers basedon haemoglobin structure (HbOC) havebeen introduced (32). This when ap-plied in concentrations up to 50 g hae-moglobin/L create optical interferencesnearly indistinguishable from those cau-sed by natural haemoglobin.

● Interference by intracellular consti-tuents with the reaction mechanism ofthe assay (chemical, biochemical andimmunological interference). In this

Fig. 33-1 Plasma samples with

various degrees ofhaemolysis

lactate dehydrogenase

potassiumcreatine kinase

triglyceridescholesterolurea, chloride,magnesium, sodium

γ−glutamyl transferase

alkaline phosphatase, amylase

aspartate aminotransferase

HDL-cholesterolalanine aminotransferase

1 2 3 4 50

4.03.53.02.52.01.51.41.31.21.11.00.9

0.5

0.80.70.6

0

haemoglobin conc. g/L

rela

tive

conc

entra

tion

(acti

vity)

Blood consists of cells and plasma.Many constituents measured in plasmahave relatively high concentrations inblood cells (233). Therefore, haemoly-sis should be avoided to obtain reliableresults.

What is haemolysis?

Haemolysis has been defined as the”release of blood cell constituents intoplasma/serum“. It is usually recognizedby a more or less reddish appearanceof the plasma/serum after centrifugation(Fig. 33-1), caused by haemoglobinreleased from the erythrocytes. As such,interference can occur even at lowerconcentrations of haemoglobin other-wise invisible to the naked eye.

The absence of redcolour does not, how-ever, exclude interfer-ence by haemolysis,

because haemoglobin isseen with the naked eye

only at a level approx-imating 300 mg/L and

higher.

Fig. 33-2 Changes in various

analytes with increas-ing haemolysis in a

dual wavelengthroutine analyzer

81

Effects of haemolysis

case, a method-dependent interferenceis observed which is not due to opticalinterference by haemoglobin. Thus,adenylate kinase released from bloodcells interferes with most standardmethods for the measurement of creatinekinase activity, the interference beingdependent on the concentration of theinhibitors of adenylate kinase added tothe reagent mixture (210). Fig. 33-3illustrates the method-dependence ofhaemoglobin interference with variousdiazo methods for bilirubin, caused bythe peroxidative effect of the haeme(224).

How to ”diagnose“, prevent and”treat“ interference brought aboutby haemolysis (73 and Annex)Overt haemolysis is easily detectedwhen the sample is visually controlledbefore being analyzed. In-vivo and in-vitro haemolysis may be differentiatedby comparing various samples of thesame patient and by analysis of sensitivemarkers of in-vivo haemolysis such ashaptoglobin and consideration of clinicalinformation. Consulting the clinician is advisable for any suspected in-vivohaemolysis. In addition any unexpect-ed increase in ”sensitive“ analytesshould be regarded as arising from in-vitro haemolysis unless this can be ex-cluded. Free haemoglobin, lactate dehy-drogenase (LDH) activity and potassiumshould increase in parallel in this case.Once diagnosed, results obtained froma haemolytic sample should be with-held (or sample not measured) if inter-ference is to be expected. If a newsample cannot be obtained, the clini-cian should receive information aboutthe possible degree of interferencealong with the result. A correction for-mula as suggested by Caraway (33)can be applied only if in-vitro haemoly-sis with parallel release of all con-

stituents can be ascertained. Evaluationof the possible cause of haemolysis willcertainly help to prevent interference.In-vitro haemolysis can be prevented bystandardizing the preanalytical phase.Use of standardized needles, closedtubes and calibrated centrifuges is ofgreat help in reducing haemolysis. Theuse of plasma instead of serum canalso minimize haemolysis, especiallyby avoiding the release of cellularconstituents from platelets (121). Fig.13-2 (see p. 32) shows how differencesbetween serum and plasma potassiumconcentrations depend on the numberof platelets.The laboratory should be aware of theeffects of haemolysis on specific tests(201). The user should expect newreagents and kits to be tested for the ef-fect of haemolysis by the manufacturerand respective information given in theproduct application manual.Every laboratory should document howhaemolyzed samples are to be handledin their quality assurance manual. Theresponsibility of the laboratory fordiagnostically reliable results can onlybe fulfilled by taking rigorous measuresto prevent misinterpretation of resultscaused by haemolysis (73 and Annex).

Fig. 33-3Interference of haemo-globin with variousdiazo methods tomeasure bilirubin(from (224))

total bilirubin fraction found in bilirubin assays studied

1.0 2.5 4.0 5.00

1.50

1.40

1.30

1.20

1.10

1.00

0.90

0.80

0.70

0.60

0.50

0.40

0.30

2,5-dichlorophenyldiazonium,detergent

2,5-dichlorophenyldiazonium,detergent

direct reading method

Jendrassik-Grof, Nosslin

Jendrassik-Grof (+Fehling)

2,4-dichloraniline2,5-dichlorophenyldiazoniumnitrophenyldiazonium

haemoglobin g/L

Does the laboratory have to know all my drugs?

82

Mechanisms of drug interferenceInterference with laboratory tests bydrugs is so widespread due to the multi-plicity of drugs and laboratory pro-cedures that a computerized directoryis the best source of information avail-able on the subject. In the main, how-ever, drug interference on laboratorytests can be broadly categorized as be-ing either biological or chemical. Apharmacological effect arises as a con-sequence of the drug being metabo-lized in the body and the metabolitesubsequently interfering with the labo-ratory test. Thus, while the parent drugpropranolol does not interfere with thebilirubin methods of both Jendrassik-Grof and Evelyn Malloy, the metabolite4-hydroxy propranolol interferes with

the bilirubin measurement by both theabove methods (208).

Another pharmacological effect relatesto the ability of the drug to increase thelevel of binding proteins. This can resultin an increase in the level of analytesbound to such proteins. For instance,oral contraceptives increase the plasmaconcentration of thyroxine-bindingglobulin, ceruloplasmin, transferrin andtranscortin, thus increasing the level ofanalytes bound (thyroxine, copper, ironand cortisol) (241).

Drug interference that is classified astechnical, on the other hand, relates toin-vitro interference which could beeither chemical or physical, such as

Tab. 34- Drug effects and interference; mechanisms and analyte changes

Category Mechanism Example Analytes Changeof drug in plasma

Enzyme induction phenytoin γ-glutamyl transferase

Enzyme inhibition in liver allopurinol uric acid

Enzyme inhibition in plasma cyclophosphamid cholinesterase

Increased oral contraceptives copper binding protein (ceruloplasmin)

Competing with novobiocin bilirubin,endogeneous com- unconjugatedponent for glucuronidation

Antivitamin warfarin, protein Ceffect phenprocoumon prothrombin

Cytotoxicity liver biguanides lactate,kidney gentamicin alanine aminotransferase

cis-platinum creatinine

Cross-reactivity spironolactone digoxin apparrentin immunoassays

Chemical reaction cephalotin creatininewith Jaffe’ reagent

Production of atypical salicylates haemoglobin A1

haemoglobins➚

➚

➚

➚

➚

➚

➚

➚

➚

➚

➚

➚

➚

1

Biologicalinfluencein vivo

Chemical andphysicalinterferencein vitro

83

Mechanisms and treatment of drug interference

haemolyzed or icteric samples etc.(141). Tab. 34- lists some of the othertypes of interference by the more com-mon drugs on laboratory tests (98,123, 141, 145, 241).

Binding of drugs to protein

The binding of drugs to proteins can bealtered due either to the presence ofother drugs competing for the samebinding site on protein or due to anincreased level of fatty acids (141). Ingeneral, drugs that are weakly boundtend to be displaced from their proteinbinding sites by a competing drug orfatty acid. Thus, free drug levels can beincreased under such circumstances.Furthermore, unless the displaced drugis rapidly metabolized, the patientwould become toxic at the therapeuticlevel of dosage.

Low albumin levels found in patientswith liver and kidney disease affectprotein binding. In such a case whenmultiple drugs are being administered,competition for binding sites on albumincan be significant. For instance, whenvalproic acid is co-administered withphenytoin, the competition for proteinbinding sites can lead to the displace-ment of phenytoin by valproic acidleading to decreased total phenytoinlevels, since the displaced phenytoin israpidly transformed into an inactivemetabolite (136).

The protein binding capacity of a par-ticular drug can be dramatically al-tered in conditions such as in uremiawhere for instance the protein bindingof phenytoin can vary from 70% tonear 0%.

The broad spectrum of drug interferencewith laboratory tests has been compiledin several reviews and books (123,

1179, 218, 241). When using these lists, the method-dependence of manyof the effects described has to be kept inmind. As with other types of interfer-ence, comparison of results obtainedusing two independent methods may in-dicate the mechanism of interference.Consulting with a clinician is advised inorder to prevent misinterpretation due todrug interference.

Sequence of sorting criteria

Dosage form Shape Colour

sugar-coatedtablet

other forms

hard gelatincapsules

soft gelatincapsules

tablet

round

triangular

oblong

oval

white

yellow

orange

red

pink

violet

blue

green

beige

brown

black

gray-silver

coloured

Fig. 34-1Colour chart of drugsused to characterizeand identify drugsfound

Everything under control?

84

Usually, the quality of a laboratory testresult is assessed by defining the impre-cision and accuracy in comparison toquality standards. These standards areeither obtained from experts, formulat-ed through one‘s own experiences ordefined by external quality controlagencies (46, 221). No such standardsexist for defining the quality of the pre-analytical phase. From present knowl-edge however, several criteria may bederived which may be taken as qualitycriteria in each individual laboratory.

Defining quality

The aim of analyzing samples takenfrom patients is to obtain a result whichdescribes the condition of the patientas represented by the analyte concen-tration in blood or other body fluids.This result helps the doctor to arrive ata diagnosis, providing it reaches himbefore he has to make a decisionwhich may well be influenced by theresult. Thus, the adequateness of therequest, the type of sample and the tim-ing has to be defined in relation to the

individual needs in any given clinicalsituation. In contrast to the analyticalresult, which is often defined as a “product“, the preanalytical phase maybe defined as a mixture of processesand materials. Tab. 35- summarizesnumerous examples of materials andprocedures (processes) used during thepreanalytical phase.

Who defines quality?

Complaints, suggestions and proposalsfrom persons involved are the majorsources of information upon which aquality assurance program for the pre-analytical phase can be initiated. Thequality of the products and processeshave to be defined in relation to themedical needs. This can be done by agroup of experts in a “quality circle“ orby an external audit. Local, national orinternational standards and recommen-dations may also be used. The results ofthese activities are published in the pre-liminary quality manual, which, afterevaluation, can be used as the basis forfuture quality assessment schemes. The

1

Tab. 35- Preanalytical processes and materials that can be subjected to quality assurance

Step Process Materials

Patient preparation Information on diet, posture and sampling procedures Urine containersPreparation of sampling Defining request, entering request, labeling tube Request form, request

program, patient and sampleidentification system

Sampling Identification of patient, timing, tourniquet, Needles, tubes, disinfectantcleaning site of sampling, selection of vein, artery or capillary sampling site, positioning of needle,changing tubes

Transport Collecting samples, Sample containers, pneumatictransporting samples tube system, cooling systems.

Sample treatment Registration, centrifugation, distribution, Identification and registrationmixing, identification, extraction program

Storage Timing of storage, selection of site Storage device, freezing and temperature, finishing storage, remixing device, temperature controlafter storage

1

85

Quality assurance in the preanalytical phase

results of this evaluation and the objec-tives of the quality assurance programshould be defined in a quality manual.

Quality of the sample

The adequateness of a sample shouldbe addressed from the standpoint ofpatient, doctor and the laboratory.

Patient’s criteria: Painless sampling ispreferable to painful sampling. Lessblood is better than excessive bloodcollection. Recently a formula was re-commended defining the optimal vol-ume of sample considering technicaland biological variables. If these rulesare followed, the minimal amount ofsample can be assured for the analysisordered (see (71) and Annex).A rapid procedure of sampling is betterthan a drawn out procedure (i.e. spot orrandom urine sample versus 24 h urineexcept where it is absolutely necessary).

Doctor’s criteria: More information fromone sample is preferable. Rapid sam-pling is preferred to time-consumingsampling. A rapid test is preferable to aslow one. The ideal sampling procedu-re is one presenting a low degree ofrisk to both patient and phlebotomist.

Laboratory’s criteria: It is better to havemore sample than is needed ratherthan an insufficient amount. An ad-equate amount has to be defined. Anormal sample is preferred to a samplewith potentially interfering (haemolytic,lipemic) or risky (infectious) factors.Standard sample size and anticoagu-lant to blood ratio is preferred to non-standardized samples.

Quality of timing

The preanalytical phase takes moretime than the analytical phase. There-

fore, it‘s timing is of critical importancefor the entire diagnostic procedure.

Fig. 35-1a gives an example of theprelaboratory and intralaboratory pre-analytical times analyzed (62, 70). Fig.35-1b shows that many different per-sons are involved; these have to be con-sidered when quality is to be improved.

How can timing be documentedand controlled?In order to clearly document the timingof laboratory testing, three essential times need to be monitored in assess-ing quality:1. Time of sampling

Fig. 35-1aRelative contributionof the preanalyticalphase to the total turn-around time ofa diagnostic test

Fig. 35-1bPersons involved inthe preanalyticalphase

▲

▲▲

Everything under control?

86

2. Time of arrival of sample in the laboratory

3. Time of printing of result

The difference between time 1 and 2gives the prelaboratory preanalyticaltime; the time interval between 2 and 3provides the intralaboratory preanalyti-cal plus analytical and postanalyticaltime. Further documentation of the vari-ous phases of the preanalytical time ispossible if analytical time is subtracted.By doing this, preanalytical phase wasshown to be responsible for more than50% of the total turn-around time inmost laboratories. Fig. 35-2 gives an

example of the documentation of pre-analytical times in a laboratory infor-mation system.

The quality journal of thepreanalytical phaseProcedures and standards used in theindividual laboratory should be docu-mented in a quality manual accessibleto all employees as well as visitors andexternal quality managers. Tab. 35-gives an example of the possible tableof contents of such a quality manual.According to the ISO standard on quali-ty management of medical laboratoriesthe quality manual has to contain a de-tailed description on responsibilities, pro-

2

cedures and aims of several preanalyticalaspects of the laboratory, including con-sequences in cases of non compliance.This list also includes procedures on re-quests, sampling and handling details aswell as regulations for transport to otherlaboratories (90).A Working Group on PreanalyticalQuality has published recommendati-ons which can serve as a basis to de-fine quality standards (71, 72, Annex).

Tab. 35- Contents of the quality manual forthe preanalytical phase

1. Request procedure a. Formsb. Patient identification

2. SamplingMaterialsa. Needlesb. Tubesc. Containers for non-blood samplesProceduresa. Venous bloodb. Capillary bloodc. Arterial bloodd. Timed urinee. Spot urinef. Cerebrospinal fluidg. Sputumh. Ascites and pleural fluidi. Other samples

3. Transport4. Registration5. Centrifugation6. Sample identification7. Storage8. Handling of interference

a. Haemolysisb. Lipemiac. Icterusd. Drugs and containments

9. Disposal of samples10.Timing of the preanalytical phase11.Documentation12.Responsibilities

2Fig. 35-2Documentation of pre-analytical times during

a usual working day

It is to be hoped that this book willhelp you to reach the gold stand-ard in the preanalytical phase.

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97

Glossary

The definition of terms used in thisvolume follows the “Vocabulary ofReference Method Procedures and Ma-terials in Laboratory Medicine”, pre-pared by R. Dybkaer (47). Othersources are given in brackets (26, 68,90, 100, 149, 156, 229).

accession:All steps necessary to ensure that a spe-cific blood specimen and theaccompanying forms are unmistak-ablyidentified as referring to a specific per-son.

accuracy:Closeness of the agreement betweenthe result of a measurement and thetrue value of the measurand.

additive:Substance, other than surface treatmentdesigned to be irremovable, that isplaced inside the receptacle to facili-tate the preservation of the specimen,or is intended to react with the speci-men, in order to allow the intendedanalysis to be performed (89).

analyte:Component of a sample indicated inthe name of a measurable quantity.

analytical interference: Systematic error of measurement cau-sed by an analytical interferent (90,149).

analytical portion: The portion of material taken from theanalytical sample and on which themeasurement of the appropriate mea-surable quantity is actually carried out.

analytical sample:A sample prepared from the laboratorysample and from which analytical por-tions may be taken.

analytical sensitivity: The slope of the analytical calibrationfunction. The term “analytical sensitivi-ty” is not a synonym for detection limit.

analytical specificity: The ability of a measurement pro-cedure to determine solely the measur-able quantity it purports to measure.

bias of measurement:Bias is the difference between theexpectation of the results of measure-ment and the true value of the measur-and.

biological influence quantity: See influence factor.

biological reference value; reference value:Value of a measurement in an individ-ual belonging to a defined referencesample group of individuals.

The term “biological reference value” isnot a synonym of the ambiguous term“normal value” as the individuals of thereference sample group may sufferfrom a defined state of disease.

component:Definable part of a system. In analytical chemistry the componentsof a system are sometimes divided into“analyte”, “concomitants”, and“solvent”; the latter two are often called“matrix”.

detection limit; limit of detection:The minimum detectable value. Resultof a measurement by given measure-ment procedure for which the prob-ability of an analytically false negativeresult is b, given the probability a of ananalytically false positive result (IUPACrecommends default values for a and bequal to 0.05.)


Glossary

98

diagnostic sensitivity: See nosographic sensitivity.

diagnostic specificity: See nosographic specificity.

endogeneous:Any factor or mechanism acting or de-rived from the system from which theanalytical sample is taken. See also in-terference factor.

error of measurement; error: Result of a measurement minus a truevalue of the measurand.

exogeneous:Any factor or mechanism added to thesample either in vivo (i.e. drug) or tothe sample in vitro (i.e. contaminant).

good laboratory practice; GLP: Organization process and the condi-tions under which laboratory studiesare planned, performed, monitored,recorded, and reported.

imprecision of measurements; imprecision:Dispersion of independent results ofmeasurements obtained under speci-fied conditions.

Imprecision of measurements, when ap-plied to sets of results of measurements,depends solely on the dispersion ofrandom error of measurement anddoes not relate to a true value of themeasurable quantity. Imprecision is usu-ally expressed numerically as the re-peatability standard deviation, an inter-mediate precision standard deviation,or a reproducibility standard deviationof results of measurements.

inaccuracy of measurement; inaccuracy:Discrepancy between the result ofmeasurement and the true value of a

measurement. Inaccuracy of a meas-urement describes a combination ofsystematic effects and random effectsthat contribute individual componentsof error of measurement.

influence, influence factor: Biological (in vivo and in vitro) influ-ence on the concentration of a mea-surand in a system (i.e. venous blood).

interference: Systematic error of measurement cau-sed by a sample component whichdoes not by itself produce a signal inthe measuring system (CEN). The effectof a substance upon any step in the de-termination of the concentration or cat-alytic activity of the analyte.

interference factor:Substance or component of the matrixof a sample which differs from the ana-lyte and interfering with the analyticalprocedure to give a false measuringsignal.

Interference factor is called influencequantity by Dybkaer (47), defined asthe measurable quantity that is not themeasurand, but that effects the result ofthe measurement.

inter-individual variation: Distribution of the values within indi-viduals of a given set.

internal quality control: Operational techniques and activitieswithin a production site that are used tofulfil requirements for quality.

international system of units; SI: Coherent system of units of measure-ment adopted and recommended bythe General Conference on Weightsand Measures (CGPM). The SI is pre-sently based on seven base units ofmeasurements: metre (m), kilogram

99

Glossary

(kg), second (s), ampere (A), kelvin (K),mole (mol), and candela (cd) (229).

intra-individual variation: Distribution of the values in a given in-dividual. It is usually assumed that thevariation occurs with time as an inde-pendent variable.

matrix:All components of the material systemexcept the analyte.

matrix effect: Influence of a sample property, otherthan the measurand, on the measure-ment and thereby on the value of themeasurand.

measurable quantity; quantity: Attribute of a phenomenon, body, orsubstance that may be distinguishedqualitatively and determined quantita-tively.

measurand:measurable quantity subject to mea-surement.

measurement:Set of operations having the object ofdetermining a value of a measurablequantity.

measurement procedure: Set of operations described specifical-ly, used in the performance of measure-ments according to a given method ofmeasurement.

metrology:Science of measurement.

nosographic sensitivity; not diagnostic sensitivity: Number of persons correctly classifiedby the results of measurement being ina defined state divided by the numberof all persons in that state.

Clinically true positive classifications di-vided by the sum of clinically true posi-tive class plus clinically false negativeclassifications.

nosographic specificity; not diagnostic specificity: Number of persons correctly classifiedby the results of measurement as notbeing in a defined state divided by thenumber of all persons not in the de-fined state.

Clinically true negative classificationsdivided by the sum of clinically truenegative class plus clinically false posi-tive classifications.

patient sample (specimen): An aliquot of a specimen that has beenappropriately collected, transportedand processed in the laboratory to pro-vide material for a specific laboratorytest (153 – 159).

preanalytical phase: synonymous with pre-metrologicalphase and pre-examination phase,starting from the request, including theexamination requisition, preparation ofthe patient, collection of the primarysample, transportation to and withinthe laboratory, and ending when theanalytical examination procedurestarts. Procedures included herein arecalled pre-examination procedures(89).

precision of measurements;precision:Closeness of agreement between inde-pendent results of measurements ob-tained under stipulated conditions (ISO3534-1).

quality assurance: All the planned and systematic activi-ties implemented within the quality sys-tem, and demonstrated as needed, to

Glossary

100

provide adequate confidence that anentity will fulfil requirements for quality(ISO 8402-3.5). In the clinical labora-tory sciences, it is customary to consid-er internal quality control and externalquality assessment as complementarybut not complete parts of quality assur-ance. The term “external quality assur-ance” is also being used to comprisethe actions ensuring the transferabilityof results of measurement.

quality system:Organisational structure, procedures,processes, and resources needed to im-plement quality management (ISO8402-3.6).

quantity:see measurable quantity.

random error of measurement; random error:Result of a measurement minus themean that would result from an infinitenumber of measurements of the samemeasurand carried out under repeata-bility conditions.

reagent:Substance employed to produce achemical reaction in order to measurequantities, pertaining to other sub-stances or convert one substance intoanother.

receptacle:Vessel, wether evacuated or not, in-tended to contain a specimen, togetherwith any receptacle accessory and ad-ditive, with closure in place.Receptacle accessory is a componentinside the receptacle which is intend-ed to assist in the collection, or mix-ing, or separation, of the specimen(89).

reference interval, reference values:Do not use normal values because ofthe inherent ambiguity of the word“normal”. The reference interval defi-nes the 95% range of reference valuesobtained from a reference population(199).

reference limit: Upper or lower limit of referenceinterval, not identical with clinicaldecision limit.

reference material: Material or substance, one or more ofwhose property values are sufficientlyhomogenous and well established tobe used for the calibration of a mea-suring system, the assessment of a mea-surement procedure, or for assigningvalues to materials (ISO-Guide 30-2.1).

reference population, reference individual: Reference individuals are personsselected by inclusion and exclusion cri-teria from a healthy population to formthe reference populations from whichreference values are obtained for com-parison with an individual having aspecific disease. The reference popula-tion should be as similar as possible tothe persons tested except for the dis-ease being investigated.

repeatability of results ofmeasurements; repeatability: Closeness of agreement between theresults of successive measurements ofthe same measurand carried out underrepeatability conditions.

sample:One or more parts taken from a systemand intended to provide information onthe system, often to serve as a basis fora decision on the system or its produc-tion.

101

Glossary

A portion taken from a system issometimes called a ”specimen”.It may be useful to distinguish be-tween ”primary sample” (taken fromthe original system), ”laboratory sam-ple” (as received by the laboratory),and ”analytical sample” from whichthe ”analytical portion” is taken.See also analytical sample, patientsample (specimen).

sampling:Process of drawing or constituting sam-ples, usually qualified by a description ofthe sampling procedure (ISO 3534-2).

sampling procedure: Operational requirements and/or in-structions relating to the use of parti-cular sampling plan, that is the plannedprocedure of selection, withdrawal,and preparation of one or more sam-ples from an inspection lot to yieldknowledge of the characteristics of thelot (ISO 3534-2).Note: In laboratory medicine, the inspection lot usually is a person.

specimen:Biological material which is obtainedin order to detect properties or to mea-sure one or more quantities (89).

stability:Ability of a system, when kept underspecificed conditions, to maintain astated property value within specifiedlimits for a specified period of time.

sterility:Sterility is the absence of living organ-isms.

systematic error of measurement;systematic error: Means that would result from an infinitenumber of measurements of the samemeasurand carried out under repea-tability conditions minus a true value ofthe measurand.

traceability:Ability to trace the history, applicationor location of that which is under con-sideration (CEN-ISO 9000: 2000).Property of the result of a measurementor the value of a standard whereby itcan be related to stated references,usually national or international stan-dards, through an unbroken chain ofcomparisons all having stated uncer-tainties (VIM; 1993, 6.10).

turn-around-time: Time interval between collection of pri-mary sample (patient sample) and re-port to the ordering health service, orTime interval between receive of re-quest and report to the requestinghealth service (90).

unspecificity:Effects of sample components otherthan the analyte that by themselves pro-duce a signal of the measuring system.

unit of measurement; unit:Particular measurable quantity, definedand adopted by convention, withwhich other measurable quantities ofthe same kind are compared in orderto express their magnitudes relative tothat quantity.

value of a measurable quantity;value:Magnitude of a measurable quantitygenerally expressed as a unit of mea-surement multiplied by a number.

venipuncture:All steps involved in obtaining anappropriately identified blood spe-cimen from a person’s vein.

Index

102 Index

Aacetate 13acetoacetate 8N-acetyl-procainamide (NAPA) 70acid citrate dextrose (ACD) 35, 61, 62acid phosphatase 7, 32, 42ACTH (see corticotropin) activated partial thromboplastin time

(APTT) 17, 52–53acute phase proteins 7addictive drugs 13additives 34 –35adenylate kinase 81ADH (see vasopressin) adrenaline (see epinephrine)age 6air temperature 10alanine aminotransferase (ALT) 8 –9,

13, 19, 36–37, 80, 82albumin 7–9, 17, 18–19, 27, 30,

32, 37, 83alcohol (ethanol) 13,aldosterone 12–13, 14–15, 17, 18alkaline phosphatase 6–7, 9, 18–19,

32, 37, 76allopurinol 82altitude 11amikacin 70amino acids 7amiodaron 70amitriptiline 71ammonia 7, 9, 32–33, 36ammonia excretion 29amphetamine 13amylase 6, 13, 16, 79, 80analyte absorption to the vessel wall

28analytical time 85 – 86angiotensin 17angiotensin converting enzyme 12antibodies 37, 78 – 79anticoagulant 21, 32, Annexantiphospholipid antibodies 79apolipoprotein AI 7, 18, 41apolipoprotein B 8, 18, 41aprotinin 53, 59arterial puncture 20–21arterialization of the puncture site 23aspartate aminotransferase (AST)

7–9, 13, 32–33, 18–19, 37, 80“atraumatic“ pencil shaped needle

26

atrial natriuretic peptide (ANP) 13, 18autoantibodies 79

Bbacteria 72bacteriological testing 72 – 75bar-code 24–25bed rest 18bicarbonate 8, 13, 57biguanides 82bilirubin 6 –9, 13, 16, 19, 32, 37,

40, 81, 82biohazard 38biological halflife 69biological influences 5, 13, 82biopsies 16blank 80blood cell lysis 62blood cells 54 –55, 60, 62– 65blood collection 60 – 61blood culture 21blood gas analysis 21, 23blood gases 21, 66 – 67blood grouping 16, 50 – 51blood smear 37, 55blood transfusion 50 –51body temperature 15borate serine EDTA 36bronchoalveolar lavage (BAL) 73

Ccadmium 12calcium (free or total) 8 –9, 18–19,

28–30, 37, 40cannabis 13capillary sampling 22–23, 66, 84carbamazepine 70carbohydrate deficient transferins 13carbondioxide (pCO2) 13, 66–67carboxyhemoglobin (CO-Hb) 12carcino embrionic antigen (CEA) 12β-carotinoids 12cartesian robot 45casts 28catecholamines 17, 30, 59cation interference 33cellular analysis 61cell separation tube 60– 61centrifugation 33, 42–43, 76, 84, Annexcephalotin 82cerebrospinal fluid (CSF) 26 –27, 69

cerebrospinal fluid (CSF) cytology 27ceruloplasmin 7, 82chemical waste 48– 49chlorazepam 69chloride 19, 29, 57, 66, 77, 80cholecalciferol (25-OH) 14cholecystokinin-pancreozymin 59cholesterol 7–8, 12–13,16–17, 18, 80cholinesterase 7, 9, 32, 82chronobiological influence 14 –15chylomicronemia 15chylomicrons 57, 76cigarettes 12circadian rhythm 14 –15cis-platinum 82citrate 16, 30, 34, 52–53, 55citrate buffered 52, 60citric acid/theophilline/adenosine/

dipyridanole (CTAD) 55clinical chemistry 56–57clonazepan 70clot activators 20clot formation 32clotting factors 7, 32caffeine 12coagulation 52– 53cocaine 69coffee 12cold agglutination 78color codes 34 – 35competition for binding sites 83complement activation 50, 59complement components 59condensation 40contamination 16–17, 69contamination control in molecular

biology 64– 65contamination with the anticoagulant

21conveyer belt system 45copper 7, 12, 78corrosive chemicals 48– 49corticotropin (ACTH) 9, 15, 58cortisol 9, 13, 14–15, 17, 58, 82cotinine 12CO2 57, 66C-peptide 58, 59C-reactive protein (CRP) 11, 16creatine kinase 6–10, 19, 32, 40,

77, 80creatinine 2–3, 7–9, 16, 19, 28,

29–30, 37, 57, 77, 80


Index 103

creatinine clearance 7, 11cristals in urine 30cryoglobulins 78cyclic AMP 12cyclophospamid 82ciclosporine A (CsA) 69, 71cylindrical robot 45cytotoxicity 82

Ddate of birth 24D-dimer 53deproteinization 56desipramine 71detergents 28, 63dextran 16diabetes mellitus 2diagnostic and therapeutic interven-

tion 16diagnostic and therapeutic proce-

dures 14–15, 16–17diet 8, 84diethylpyrocarbonate (DEPC) 64digitoxin 68, 71digoxin 68, 71, 82digoxin-like immunoreactive

substances 69disopyramide 70direct bilirubin 32direct potentiometry 56disposal 46 – 49disposal of chemicals 48 – 49disposal of needles 46, 48disposal of specim 47– 48disposal of sharp objects 46 – 47distribution of sample 42– 43, 84diurnal changes 58diurnal variation 14–15DNA (desoxyribonuclein acid)

62– 65documentation 85 – 86dopamine 32drinking 8drug colour chart 83drug effects and interferences 82–83drug monitoring 15, 31, 68 –71drugs 68 –71, 82 – 83“dry chemistry“ 56duration of storage 36

Eedema formation 18EDTA (ethylenediaminetetraacetic-

acid) 2, 34, 50, 54 – 55, 58 – 59,61, 62, 68 – 69

EDTA-aprotinin mixture 53EDTA dependent antibodies 78EDTA plasma 58EGTA 59effect of light 40electrolytes 56, 57, 66elimination half-life of drugs 70 –71endorphin 58β-endorphin 58enzyme induction 82eosinophils 15epinephrine 9, 12–13, 15, 18ergometry 16European standard on sample trans-

portation 39erythocyte sedimetation rate 7erythocytes 7, 18–19, 37, 78estriol 11estradiol 13ethanol 13ethosuximide 70evaporation 40exercise 9–10, 14, 16, 19explosive chemicals and waste

48 – 49extraction 84

Ffactor VII 53factor VIII 53factor XI 53factor XII 53femoral artery 21ferritin 7fibrin degradation products (FDP) 53fibrin monomers 17, 41fibrinogen 12, 16–17, 32– 33, 53fibrinolysis 53FICOLL-HYPAQUE 60 – 61finger puncture 22–23first morning urine 28flammable waste 48 – 49flow cytometry applications 61fluoride 26, 34 – 35folate 13folic acid 40follitropin (FSH) 58food depriviation 8food intake 8free fatty acids 8, 13free hemoglobin 81freezing of blood 69fructose 16fungi 73 – 74

Ggas diffusion 40gas exchange 67gastrin 58gastro-inestinal tract surgery 16gel barrier 60–61gender 7gene rearrangements 62genes 62– 65gentamicin 70, 82γ-globulin 16, 33glas capillaries 67glas syringes 67, 69glomerular filtration rate 7glucagon 8–9, 59glucose 2–3, 7–9, 12–13, 16–17,

19, 21, 30, 36, 40, 57, 77glucose tolerance 8, 14glutamate dehydrogenase 13glutamine hydrolysis 32γ-glutamyltransferase 7, 9, 13, 19,

32, 37, 80, 82glutathione 59glycerol 8, 12glycolysis 40, 67glycolytic inhibitors 21, 34 – 35glycoproteins IIIA, IIb 79gold standard 5, 87granulocyte lysis 80granulocytes 6, 61granulocyte viablity 61growth hormone 58guanidinium isothiocyanate 63gut glucagon 58

Hhaematin 62haematocrit 9, 11, 12, 18, 37, 52,

56haematology 54 – 55haemoglobin 6–7, 11, 15, 17,

18–19, 37, 80 – 81haemoglobin A1c 3–4, 82haemolysis 33, 50, 80 – 81, Annexhaemostasiology 52– 53hazardous waste 48– 49HDL-cholesterol 6 –7, 12, 18, 80heavy metals 12heparin 34, 58 – 59, 60 – 61, 62, 66,

68heparin interference 33, 62heroin 13heterophilic antibodies 79high fat diet 8

104 Index

human leucocyte antigen (HLAtyping) 60

hydrochloric acid (HCL) 305-hydroxy indolacetic acid 30hydroxyproline in urine 7hydroxybutyrate 8hydrolysis of glutamine 32hyperlipidemia 76 – 77hypotonic lysis 61hypoxia 10

Iicteric sample Annexidentification of patient 20, 50, 84identity of samples 24 –25, 41imipramine 71immune hematology 50 –51immunoassays 59immunocytology 27immunoglobulin A 18immunoglobulin G (IgG) 7, 18, 27, 41immunoglobulin M 18indirect sample identification 25infectious substance label 38influence of temperature on serum

analytes 36information flow 24infusion site 17infusions 16 –17injections 16inorganic phosphate 8 –9, 16, 19,

29–30, 32, 36, 42insulin 8–9, 13, 16–17, 58interference 33, 76 – 77, 80 – 81interference factors 5iodoacetate 35ionising radiation 16ionized calcium 66–67ion release from blood cells 67iron 7, 15, 32, 82isometric exercise 9isopropanol 22isotonic exercise 9

Jjointed robot 45

Kketone bodies 8–10

Llabeling 80laboratory computer 24–25

lactate 8, 10, 13, 17, 19, 21, 27,32, 40, 67, 82

lactate dehydrogenase (LDH) 19,32–33, 37, 42, 43, 80

LDL-cholesterol 6 –7, 12, 18, 41lead 12leucocytes 18–19, 27, 37, 78 – 79leupeptin 59lidocaine 70light effects 40lipase 13lipemia 76 – 77, Annexlipemic sample 76 – 77, Annexlipids 57lipoprotein(a) 12lipoprotein electrophoresis 41lipoprotein X 41lithium 32, 71long term starvation 8lutropin (LH) 58lymphocyte separation 60–61lymphocytes 6, 12, 60, 78lysis of red blood cells 62lysis of the cells 32

Mmacroamylase 79macro creatinine kinase (CK) 79macroenzymes 79magnesium 29–30magnesium phosphate 41mailing of blood 36–37, 38 –39malnutrition 8marathon race 9material safety data sheets (MSDS)

49Mean cellular (corpuscalar) volume

(MCV) 12–13, 37, 54, 78meal 8, 15menstrual cycle 14mental stress 16 –17metabisulfite 59metabolic acidosis 13metabolism of blood cells 67method-dependent interference 33, 81methotrexate 71microbiology testing 72 – 75microbiology in CSF 26–27, 73micro blood collection sets (disposal)

47– 48microcollection technique 23microcollection tubes 42– 43mid-stream urine 29mitochondrial DNA 62mixing 55, 67, 84

mixing bar (“flea“) 66mixing of deep-frozen samples 41mixing of specimen 20, 67molecular biology 62– 65mononuclear cells 61monocytes 6, 12, 60–61morphine 13murine monoclonal antibodies 79muscle mass 7, 10mycological testing 73 – 74mycobacteria 29myoglobin 7

NNa+, K+-ATPase 57nafamostate mesylate 59needle disposal 46 – 48needle reshielding 47netilmicin 70neuron specific enolase 32neurotensin 13, 58neutrophils 12neutrophil morphology 54nicotine 12nitrazepam 69non-ionic detergents 63norepinephrine 9, 13, 15, 18nortryptiline 71novobiocin 82

Ooligoclonal protein bands 27operations 16optical interference 80oral contraceptives 82oral glucose tolerance test 16, 22order of collection 21osmolality 18, 30osteocalcin 13overfilling tubes 20ovulation 14oxalate 30, 68oxygen (pO2) 67

Ppancreatic peptide 58pancreatic polypeptide 13parasites 74 – 75parasitological testing 74 – 75parathyroid hormon related protein

(PTH-RP) 59patient preparation 84patient’s criteria of quality 85pepsinogen-1 58

Index 105

pepstatin 59pH 8, 21, 40, 67pharmacological effect 82phenobarbitol 70phenol 63phenprocoumon 82D-phenylalanine-proline-arginine-

chlormethylketone 53phenytoin 70, 82 – 83pH value in blood 16, 21, 66–67phlebotomy 20 –21phosphate, phosphorus 8–9, 15, 16,

22, 30, 32phosphodiesterase 12plantar surface of the heel 22plasma 16, 32– 33, 57plasma-serum differences 32– 33plasma water 56plastic syringes 67, 69plastic tubes 66plasticizers 69platelet contamination 43platelet count 32, 55platelet derived growth factor 55platelet factor IV (PF4) 55platelet free plasma 33platelet lysis 80platelet poor plasma 33platelet rich plasma 33platelet satellism 55platelets 32–33, 43, 54 –55, 60, 61,

78pneumatic tube delivery system 36polyagglutination 50 – 51polymerase chain reaction (PCR) 62,

65porpyrins 30, 40position of patient 20post-centrifugal coagulation 33posture 18, 58, 84potassium 2–4, 8 –9, 13, 15,

16–17, 19, 22, 29–30, 32–33,36–37, 42, 57, 66–67, 80 – 81

pO2 67preanalytical times 85 – 86pre-ovulatory increase 15pregnancy 7preservation of cells 35primidone 70procainamide 70proinsulin 58prolactin 7, 12–13, 15, 17protease inhibitor 59protein: see total proteinprotein binding of proteins 70–71, 83protein C 78

protein electrophoresis 33proteolytic enzyme inhibitor 58prothrombin time (PT) 17, 52- 53, 82pseudoagglutination 16pseudoleucocytosis 78pseudothrombocytopenia 55, 79pseudothrombocytosis 78proteolytic enzyme inhibitor 58 – 59pyridoxal phosphate 12pyruvate 8, 19pyruvate kinase 9

Qquality assurance 84 – 87quality managers 86quality manual 86quality of the sample 85, Annexquality of timing 85quinidine 70

Rrace 6–7radial artery 21ratio of anticoagulant to blood 52, 54red cell mass 12refrigeration 58renin 11, 13, 15, 17, 18, 58reptilase time 16–17request 84request form 24, 84reshielding of needles 47restriction fragment lenght polymor-

phism (RFLP) 62–64reticulocytes 7retinol binding protein 8rethawing effects 40 –41ribonuclease (RNAses) contamination

64rimixing after storage 41, 84RNA (ribonucleic acid) 62– 64robotics 44– 45rubber closures 69running 9–10

Ssafety aspects 46 – 49safety flow lancet 22safety labels 49salicylates 78saliva collection 31, 69sample handling 43, 55sample identification 24 – 25sample inhomogeneity 40, 72sample preparation 64, 67

sample sorter 44sample treatment 84sampling 14 –15, 26 – 27, 52, 58, 67,

68 –71sampling from catheters 17, 21satellism of platelets 55scalp artery 21scheduling infusions and blood sam-

pling 17seasonal influences 14second morning urine 28secretin 58–59selection of vein 84selenium 12serotonin 32serum 16, 32– 33serum osmolality 11serum-plasma differences 32– 33site for sampling blood 20, 22– 23skin puncture 20, 22– 23sleep 14smoking 12sodium 8–9, 13, 15, 19, 30, 32, 37,

57, 66, 77, 80sodium azide 30sodiumborate serine EDTA 36sodium carbonate 30sodium dodecylsulfate (SDS) 63somatotropin (growth hormone) 9,

15, 17somatostatin 58–59soybean trypsin inhibitor 53specimen collection 28–31, 52, 55, 66specimen collection bags 28specimen processing 42 – 43spironolactone 82sputum tube 74 – 75stability of the constituents in the sam-

ple 30, 40 – 41, 55, 69stabilising RNA 64standard meal 8starvation 8, 15, 29steady-state of drugs 70 –71stool tube 73 – 75storage of samples 27, 30, 40 – 41,

55, 57– 58, 64, 67, 69, 84, Annexstorage temperature 30, 36, 51streptokinase 53streptomycin 70subsamples 24substance P 58supine position 18surgical procedures 9, 16sustained-release preparations 70swap specimen and tube 73 – 75swimming 9

106 Index

Ttacrolimus 71Taq DNA polymerase 62temperature (body) 15temperature control 84temperature during transport 36 –37testosterone 15thawing samples 40 – 41theophylline 68, 71therapeutic drug monitoring (TDM)

68 –71thiocyanate 12thrombin 53thrombin time 16–17β-thromboglobulin 55thrombocytosis 67thromboxane A2 55thymol 30thyroid-stimulating hormone (TSH)

13, 15, 17, 32thyroxine 7, 9, 13, 15, 18, 79, 82thyroxine-binding globulin 82 time after drug 14–15time after last meal 14–15time after last sample 14–15time dependent changes 14time of sampling 14–15, 24time of the day 14–15time saving 33timing 14–15, 24, 58, 68, 84 – 85timing of storage 84, Annextobacco smoke 12tobramycin 70total bilirubin 32total protein 7, 16, 18–19, 30, 32,

77, 82

tourniquet 19, 20, 66, 84tourniquet application time 19toxic chemicals 48 – 49trace elements 57training status 9–10, 11transcortin 78transferrin 32transfusions 16 –17transport of diagnostic specimen 27,

36 – 37, 38 – 39, 53, 55, 58, 64,67, 69, 84

transport of analytes 55tricyclic antidepressants 69triglycerides 7–8, 13, 18–19, 32,

56, 57, 76 – 77, 80triiodothyronine (T3) 8, 32, 79thrombocytolysis 32 – 33thrombocytosis 67tube and sample disposal 47turbidity 15, 76 – 77turbid samples 76 – 77turn around time (TAT) 44, 85 – 86two-dimensional bar code 25two-dimensional dotcode 25

Uultracentrifugation 77umbilical artery 21upright position 18urea 7–9, 16, 19, 29–30uric acid 7–11, 13, 16, 19, 22, 30,

41, 73, 78urinary creatinine 10–11urinary excretion of calcium 18, 28urine 28 – 30, 69urine containers 28, 84

urine pH 30urine preservation 30urine sediment 41urine volume 7, 15urobilinogen 30urokinase 53

Vvalproic acid 70, 83vanillylmandelic acid (VMA) 13vancomycin 70vasoactive intestinal peptide 58vasopressin 13, 17venipuncture 20 – 21very low density lipoproteins (VLDL)

9, 76viruses 75volume displacement effect 56volume errors 33volume of blood collected 21volume shift 10, 18–19von Willebrand factor 16

Wwarfarin 82water displacement 77white blood cell differential count 54whole blood 57, 67whole blood lysis 61workflow 44, 45

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