Vol. 10 - I988 Pharmnceutisch Weekblan‘ Scientific Edition
`
`185
`
`review articles
`
`Plasma or serum in therapeutic drug monitoring and clinical toxicology
`
`DONALD ILA. USES
`
`Introduction
`
`in therapeutic drug monitoring and clinical toxi-
`cology most determinations of the concentration of a
`substance are carried out in blood or blood com-
`
`ponents. Whole blood can only be analysed if the
`blood has been sampled in collecting tubes con-
`taining an anticoagulant. When such samples are
`centrifuged, plasma is obtained. Therefore, plasma
`always contains one or more anticoagulants. Hep»
`arin sodium (3-too U/ml) is most widely used for this
`purpose, but citrate, edctate, oxalate and fluoride
`are also suitable additives. Scrum is the clear liquid
`that separates from blood when blood is allowed to
`clot completely and then centrifuged. Therefore
`serum contains no fibrogen and no anticoagulants.
`The choice between whole blood on the one hand
`
`and either plasma or serum on the other is clear.
`Whole blood concentrations are only measured if the
`compound is concentrated in the erythrocytes (cg.
`lead, cyanide, mercury, carbon monoxide, chlor-
`thalidone),"3 if there is a fluctuating erythrocyte-
`plasma ratio (ciclosporin A),“ or because of the risk
`of loss during storage or centrifugatiorfi
`The clinical effect of several compounds correlates
`better with the concentration in a tissue compart-
`ment than with the serum or plasma concentration.
`Considering erythrocytes as a readily available tis-
`sue, whole blood should he the matrix of choice in
`therapeutic drug monitoring ’ and toxicology.“
`Sometimes lymphocyte concentration can predict
`the thcraapeutic effect better (epirubicin, mitox~
`antrone). 9 In nearly all other cases, plasma or
`serum concentrations are measured.
`
`is there any difference between serum and plasma
`concentration?
`
`The expression ‘plasma concentration‘ is a part of
`the title of many articles in the literature. Reading
`these articles, however,
`it appears that serum
`samples have often been taken. The authors very
`seldom present
`their arguments for
`the choice
`between plasma or serum.
`Some of the advantages of plasma over scrum
`are:
`
`~ large volume;
`— no delayed clotting;
`— less risk of haemolysis;
`— the sample is often suitable for both whole blood
`and plasma monitoring.
`Some of the disadvantages of plasma over scrum
`are:
`
`— the (unknown) influence of the anticoagulant on
`the assay, the protein binding and the stability of
`the sample:
`- the (unknown) influence of additives or impurities
`in the anticoagulant on the assay and the concen-
`tration;
`— the risk of the formation of small clots (incomplete
`mixing, instability);
`- dilution of the sample;
`— sampling is more expensive;
`— choice of anticoagulant can be confusing.
`
`ADVANTAGES OF PLASMA OVER SERUM
`
`Larger available volume
`If blood is allowed to clot and is then centrifuged,
`
`Keywords
`Anticoagulants
`Blood coagulation
`Blood preservation
`Plasma
`Protein binding
`Serum
`
`Therapeutic drug monitoring
`Toxicology
`
`‘Laboratory for Clinical and
`Forensic Toxicology and Drug
`Monitoring, Depanment of
`Pharmacy, University Hospital
`Groningen, 13.0. Boa 3,o.oo1,
`9*,-oo RB Groningen.
`
`
`1
`
`p
`
`Uges BRA. Plasma or serum in therapeutic drug monitoring and clinical
`toxicology. Pharm ‘Wcckbl [Sci] t983;ro:i85-8.
`
`Abstract
`The relative merits of plasma and serum in blood analysis are reviewed.
`The expression ‘plasma concentration‘ is often used in the literature,
`although serum samples have been taken. In most cases serum and plasma
`concentrations of analytes are the same. The choice depends mostly on
`the policy of the hospital or the availability of the test tubes in the ward.
`Some of the advantages of plasma over serum are large volume, on
`delayed clotting, less risk of haemolysis. In addition, the sample is often
`suitable for both whole blood and plasma monitoring. Some of the
`disadvantages of plasma over serum are the (unknown) influence of the
`anticoagulant on the assay, on the protein binding and on the stability of
`the sample, the (unknown) influence of additives or impurities in the
`anticoagulants on the assay and on the concentration, the risk of the
`formation of small clots and dilution of the sample.
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`Vol. to - 1938 Phnrmaceutirch Wcekblad Scientific Edition
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`about 3:: to 50% of the original volume is collected as
`serum {upper layer). A slightly larger sample (in
`general 50%), known as plasma, can be obtained by
`centrifugation of a blood sample collected in a tube
`containing an anticoagulant. Serum and plasma are
`not significantly different with respect
`to drug
`analysis.
`Despite the fact that serum is devoid of the
`proteins associated with the clotting process, the
`most important proteins (quantitatively speaking),
`such as the albumins and globulins, are present in
`similar amounts (approximately 2% wt/vol) in both
`serum and plasma. Thus plasma is in general pre«
`ferred because of its greater yield from blood
`samples. The greater yield the greater the amount of
`drug and the fewer the problems with sensitivity, or
`with sampling neonates.” “
`
`Delay through time needed for cloning
`Clotting can sometimes be of long duration. This is
`especially true when samples are taken in polypro-
`pylene test tubes, in which case clotting can continue
`even after centrifugation.
`
`Decreased risk of heemolysis
`The chlortlialidone concentration in erythrocytes
`is about 4.0 times as high as that in plasma} One
`percent of haernolysis (which cannot easily be seen)
`will increase the apparent plasma concentration by
`25%. This problem is even more acute when serum
`samples are produced from whole blood.
`The separation by centrifugation should be carried
`out as quicldyas possible in order to prevent any
`effects due to lysis of the blood clot. " This phenom-
`enon is well known in the analysis of the endogenous
`compounds potassium and iron in serum.
`
`Suitability of the sample for both whole blood and
`plasma analysis
`If several analyses have to be carried out on the
`blood of a single patient, determinations can be
`carried out in whole blood as well as plasma from one
`blood sample containing a suitable anticoagulant. In
`bedside chemistry whole blood is preferred to
`plasma or serum, because this avoids a centri-
`fugation step.”
`
`DISADVANTAGES or PLASMA
`
`IN RELATION
`
`ro ssnuyrr
`
`The influence of the anticoagulant on the assay
`Walters and Roberts showed that heparin at a
`concentration of too U per ml interfered with the
`gentamicin EMI'"l‘® assay, but not with the Tl“)-x®
`F-‘PIA assay.” This effect is thought to be due to the
`inhibition of enzyme activity rather than disturbance
`of the antigen-«antibody reaction. Blood collected
`into heparinized tubes usually contains approxi-
`mately 30 U of heparin per ml and at this level no
`interference with the EMITW assay occurs. How-
`
`ever, plasma derived from specimens collected from
`arterial and venous blood is likely to yield erroneous
`results.
`The formation of a gentamicin—heparin complex
`has been demonstrated by Myers crol. ‘ Cipolle at all
`concluded that heparinized tubes should not be used
`when collecting blood samples for assays where
`inactivation of gentamicin would give false values. ‘5
`Ecletate was for gentamicin a suitable alternative.
`On the other hand, the Dutch foundation ‘Quality
`control
`clinical drug analysis
`and toxicology’
`(KKGT) found with its quality control programme
`that eclctate interfered strongly with the FPIA assay
`and not with the Ell/Il’I‘® assay of carbamazepine and
`with the EMIW assay and not with FPIA assay of
`valproic acid. The reason of this interference is
`unknown (Dijlthuls IC, personal communication,
`1988).
`
`The influence of the anticoagulant on protein
`binding
`A discrepancy in the measurement of lidocaine in
`heparinized blood and in serum could be expected,
`as heparin increases the free fraction of lidocaine.“
`Lidocaine is subsequently redistributed from the
`plasma into red blood cells.
`The percentage of free ibuprofen in heparinized
`plasma is significantly higher than in non—heparin-
`ized plasma. 7 Although small doses of heparin can
`affect drug binding, the extent and variability of the
`effect depends on the biological activity of the
`heparin, and varies with the manufacturer and
`batch, time of sampling, and food intake. 18 Although
`the intravenous administration of heparin results in a
`high lipolytic activity in vice and consequently
`increases the concentrations of non-esterified fatty
`acids in vitro, it is unlikely that this will happen after
`adding heparin to the tube.
`Nomesterified fatty acid can displace numerous
`drugs from their binding sites in plasma. When the
`nomesterified fatty acids concentration increases in
`virro, phcnytoin will be displaced from the binding
`sites. The free phcnytoin then shifts from the plasma
`into the erythrocytes, resulting in a lower phenytoin
`plasmalevel (up to 2o% below its original value!).”
`The same phenomenon was found with amitrip-
`tyline, imiprarnine and rnaprotiline.‘ The recovery
`of chlorprornazine added to heparinized plasma in
`vitro was reduced compared with that obtained with
`water or oxalated plasma.”
`
`The influence of the anticoagulant on the stability
`of the sample
`When heparin deteriorates or adsorbs onto the
`wall of the collecting tube, clotting will still occur in a
`clear plasma sample. The pH of the sample can be
`changed by using sodium citrate, or oxalate as
`anticoagulant. This change of pH may influence the
`stability of the drug in the sample (cg. atractuium).
`Sodium fluoride can be used as anticoagulant (cal-
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`‘Vol. 10 - I988 Pharmacsutirch Weekbiad Scientific Edition
`
`:87
`
`cium binder), and as a preservative (antimicrobial)
`of ethanol. Blood samples that are to be tested for
`ethanol must contain in mg sodium fluoride per ml
`during storage.“ 22
`
`The influence of additives or impurities’ in the
`anticoagulant on the assay
`Benzyl alcohol, which may he added to heparin as
`a preservative, can disturb the fluorimetric assay of
`corticosteroids.” Serum zine concentrations are 16%
`higher than plasma concentrations. This phenom-
`enon was explained by a release of zinc from
`platelets during coagulation of the blood sarnples.”
`Keyzer at at’. , however, found no difference between
`the serum and plasma zinc levels of fifty volun-
`tears.”
`
`The influence of additives or impurities in the
`anticoagulant on the concentration
`Anticoagulants can contain impurities which are
`precisely the compounds to be determined, e.g. lead,
`aluminium, copper, fluoride.
`
`The risk of clot formation because of poor mixing
`or poor stability
`Unfortunately, blood samples in heparinized
`tubes are sometimes not very well mixed. In such
`cases a partially clotted sample will arrive in the
`laboratory.
`
`Improper dilution of the sample
`We have had a number of incidents in which blood
`
`samples of about 5 ml were diluted with about I ml of
`a solution of anticoagulant (20% dllutionl). Differ-
`ences in serum and plasma transcohalamin 11 levels in
`the literature have been explained bet dilution by
`EDTA or sodium fluoride solutions.‘
`
`Cost
`
`Sample tubes with heparin are approximately 10%
`more expensive than non-heparinized tubes.
`
`The choice of anticoagulant
`There are several anticoagulants, each of which
`must be used in a different concentration. Examples
`are sodium heparin,
`lithium heparin, potassium
`edetate, sodium edetate, sodium citrate, and sodium
`fluoride. The differences in the influence of these
`
`anticoagulants on the various assays are largerly
`unknown.
`Differences were demonstrated between serum
`
`and plasma levels of endogenous compounds, which
`may be of sufficient magnitude to alter clinical
`interpretation of some results when using different
`radioassay procedures and different anticoagu-
`lants.“ These differences are in general larger with
`immunoassays than with chromatographic methods.
`Cholinesterase activity can be determined in serum
`or heparinized plasma. Sodium fluoride and citrate
`should not be used as anticoagulants because they
`
`depress cholinesterase activity as measured by sev-
`eral methods.”
`Anticoagulants used in sampling plasma can con-
`found diffusion assays by causing alterations in the
`agar gel matrix. Therefore, in the microbiological
`measurement of, for example, erythromycin. cytara-
`binegr 5-fluorouracil the use of serum is recommen-
`ded.
`
`Conclusion
`
`In conclusion, we can say that in most cases serum
`and plasma analyte concentrations are the same. The
`choice depends mostly on the established practice in
`the hospital or the kind of available test tubes in the
`ward. In special cases such as the determination of
`free drug levels, however,
`there are important
`differences. It is advisable, therefore, that during
`kinetic studies of drugs, the similarity of serum and
`plasma levels for the compound being studied be
`proven. Furthermore, to avoid confusion. authors
`should be careful and precise in their presentations
`so that readers are aware that blood, plasma or
`serum is the sample under discussion.
`
`References
`
`‘ Casarett LI, Doull J. Toxicology. The basic science of
`poisons. New York: Macmillan Publishing Company,
`1975-
`’ Moffat AC. ed. Clarl-:e’s isolation and identification of
`drugs. 2nd ed. London: The Fharmaceutical Press,
`1986.
`3‘ Beennann B, Ciroschinslty-Grind M. Clinical pharmaco-
`ltinetics of diuretics. Clin Pharmacolcinet 198o;5:22r-45.
`‘ Keown PA. Stiller CR. Stawecki M, Mclvlichael J,
`,1-Iowson W. Pharmacoltinetics and interactions of ciclo-
`sporin. In: Schindler R, ed. Ciclosporin in autoimmune
`diseases. Berlin: Springer-Verlag, 1985239-42.
`5 Uges DRA, Van der Paauw l~lJ'W. Alcohol, dc bleed-
`proef en de contra-expertise {Alcohol. blood test and
`counterenpertise}. Ned Forens Tijdschr I982;2:7~9.
`" Maguire KP, Burrows GD. Norman JR. Scoggings BA.
`Blood plasma distribution ratios of psychotropic drugs.
`Clin Chem I93D;26:I624-5.
`7 Linnoila M, Dorrity F, Jobson K. Plasma and erythro-
`cyte levels of tricyclic antidepressants in depressed
`patients. Am .1‘ Psychiatry1978;135:557-fit.
`3 De Vries EOE, Clreidanus J, Mulder NH. et al. A phase
`I phannacolcinetic study with 2:-clay continuous infusion
`of epirubicin. J Clin Oncol I987;5:r445-51.
`" De ‘Vries EOE. Greidanus J. Mulder NH, at al. A phase
`1 and pharmacolrinetic study with 21-day continuous
`infusion of mitoxantrone (MK). Am Soc Clin Oncol
`I98E;?:58.
`y
`‘
`“’ Smits RV, Stewart IT. Textbook of biopharmaceuttc
`analysis. Philadelphia: Lea dc Febiger, 19813-rd.
`" Toseland PA. Samples and sampling. In: Moffat AC,
`ed. Clarkes isolation and identification of drugs. 2nd
`ed. London: The Pharmaceutical Press, I986:rrr-7.
`“ Hichs IA,
`losetsohn M. Another physicians ofhce
`analyser:
`the Abbott vision evaluated. Clin Chem
`t987;33=8x7~s.
`_
`_
`*3 Walters MI, Roberts WH. Gentamicin/heparin inter-
`actions: eftects on two immunoassays and on protein
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`binding. Ther Drug Monitor :l984;6:I99~202.
`"' Meyers DR, DeFehr J, Bennett ‘WM, Porter GA, Olsen
`GD. Gentarnioin binding to serum and plasma proteins.
`Clin Pharmacol Ther 1g78;23:356»6o.
`'5 Cipolle RI, Zaslte DE, Crossing; K. Gentamioin/tobra-
`myein. Therapeutic use and serum concentration moni-
`toring. In: Taylor WI, Finn LA, eds. Individnalizing
`drug therapy. I. New York: Frank Cross Townsend Inc. ,
`I98t:i33.
`“ Dtibe LM, I-Io-Ngoc A, Davies RF, Beanlends D5.
`Mousseau N, Mcfiilveray IJ . Influence of protamine on
`liepmrine induced increases of lidoeaine free fraction.
`Res Comrmm Chem ?athol Pharmaeol
`I9B3;42:4o1-
`:5.
`" Pearce GA, Brown KP. Heat inhibition of in vitro
`Iipolysis and "C ibuprofen protein binding in plasma
`from heparinizcd nraemit: subjects. Life Sci 1983;
`33: 1457-66.
`‘"5 -Naranjo CA, Sellers EM, Eilhouw V, Alexander 1?, Fan
`T, Shaw 3. Variability in heparin effect on serum drug
`binding. Clin Pharmacol Thor ig8o;28:5,45-5o.
`” Schulz P, Abang A, Giaeomini JC, Blaschlte TF,
`Ciiaeomini KM. Effect of heparin on the red blood
`eel}-to-plasma concentration ratio of diphenylhydantoin
`and przzosin. The: Drug Monitor 1983;5:4g*;-g.
`2" Whelpton R. Tricyclio antidepressants and neuroieptios.
`In: Curry AS, ed. Antalytioai methods in human
`toxicology. Part I. Weinheim: Verlag Chemie, 1985:
`I 9«-58.
`3‘ Flanagan R1, Widdop 13. Clinical toxicology. In: Corry
`AS, ed. Analytical methods in human toxicologr. Part I.
`Weinheirn: Verlag Chemie, 198537-66.
`
`2’ Froentjes W, Schute JB, Strengers Th, Verwey AMA.
`Analytiseh-ehemische aspecten van 1216 wijziging van de
`Wegenverlceerswet (ti) {Analytical and clinical aspects
`of the change in the Traffic Act 01)}. Pharm Weelthl
`2976;: lf 1:349-62.
`2*‘ Work BE jr. Interference of heparin containing henzyl
`alcohol
`in the fluorometric determination .01’ plasma
`corticosteroids. J Clin Endocrine! r967;27:I35o.
`3" Foley B, Johnson SA, Haoltley B, Smith JCS jr, I-Ialsted
`IA. Zinc content of human platelets. Prot: Soc Exp Biol
`Med 1968;128:265-(9.
`*5 Keyzer JJ, Oosting E, ‘Woithers Bi}, Muskiet FAJ,
`Hindri ks FR. Van der Slik ‘W. Zinc in plasma and serum:
`influence of contamination due to the collection tubes.
`
`Pharm Weekbl {Sci} 1933-,;§‘,:248-51.
`3*‘ Carmel R. Vitamin B12-binding proteins in serum and
`plasma in various disorders. Effects of anticoagulants.
`Am J Pathol i978;69:31g-25.
`” Kubasilt NP, Sine HE. Results for serum and piasma
`compared in I5 seiected raciioassays. Ciin Chem
`I978;24: I37-9.
`33 Huizinga JR, Grips CH. Evaluation of the UV—34o
`spectrophotometric determination for pseudot:holin-
`esterase activity (EC 3.1.1.8) in human serum. .1 Clin
`Chem Clin Biochem I987;25:I6I-5.
`3° Smite RV, Stewart JT. Mierobiologio assay methods. In:
`Textbook. of biopharmaceutie analysia. Philadelphia:
`Lea 8: Febiger, 14381349-59.
`
`Received December 1987.
`Revised April 1988.
`Accepted May 1988.
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