accurate results in the clinical laboratory || issues of interferences in therapeutic drug...

C H A P T E R

13

Issues of Interferences inTherapeutic Drug Monitoring

Gwendolyn A. McMillin, Kamisha L. Johnson-DavisUniversity of Utah School of Medicine and ARUP Laboratories, Salt Lake City, Utah

INTRODUCTION

Therapeutic drug management (TDM) is a corner-stone of personalized medicine, designed to select andoptimize drug therapy for individual patients. By opti-mizing therapy, the desirable effects of a drug (effi-cacy) can be maximized and the undesirable effects ofa drug (toxicity) minimized. TDM is also employed toevaluate patient compliance, identify drug�drug inter-actions or other changes in pharmacokinetics, and bothcharacterize and manage acute intoxications or emer-gent overdose situations. TDM is most useful whenthere is a good correlation between drug concentrationin blood and effect (therapeutic and toxic). It is partic-ularly useful for drugs that have a narrow therapeuticwindow, prodrugs, and drugs that are highly bound toplasma proteins. TDM is not widely available for mostdrugs due in part to limitations in analytical methods.

Inaccuracies in TDM results will impact patient careby contributing to unnecessary or inappropriate doseadjustments. Inappropriate increases in dose couldlead to dose-dependent adverse drug reactions(ADRs). ADRs are reported to account for 41% of allhospital admissions, due to inappropriate dose or pre-scription, allergic reactions, and drug�drug interac-tions. The consequences of ADRs are costly and can befatal [1]. Errors in TDM results could also cause unnec-essary dose reductions and minimize the therapeuticeffect. Therapeutic failure due to inappropriately lowdosing could contribute to unnecessary testing andprocedures, and for time-sensitive conditions such asorgan transplantation, cancer, uncontrolled seizures,and cardiac disturbances, it could be fatal. The labora-tory has an important opportunity to promoteclinically useful TDM through involvement in

appropriate specimen collection, sample handling,sample processing, selection of appropriate analyticalmethods, and interpretation of results. As such, thelaboratory must be prepared to consult regarding ther-apeutic ranges, toxic thresholds, as well as strengthsand limitations of available analytical methods.Inaccuracies in TDM may be a result of pre-analyticalfactors surrounding the specimen and patient or ana-lytical factors surrounding selection and performancecharacteristics of the analytical method (Figure 13.1).Sources of interference may arise from endogenous orexogenous sources and may impact specific technolo-gies in a direct or indirect manner. However, by usinggood laboratory practice, interferences can be identi-fied, managed, and/or prevented.

This chapter describes sources of pre-analytical andanalytical interferences that have been reported toadversely affect the accuracy of TDM results. Specificdrug, technology, and case examples are provided toillustrate these sources in more detail and guideappropriate utilization and interpretation of TDMfrom the clinical laboratory perspective.

SOURCES OF PRE-ANALYTICALINTERFERENCES IN TDM

Pre-analytical sources of inaccuracy in laboratorytest results are discussed in detail elsewhere in thisbook (see Chapters 1�4). However, there are manypre-analytical factors important to TDM that areunique from other types of laboratory testing, andthese are briefly emphasized here. In particular, thecoordination of pharmacokinetic factors for the specificdrug formulation(s) of interest, with the clinical status

195Accurate Results in the Clinical Laboratory.

DOI: http://dx.doi.org/10.1016/B978-0-12-415783-5.00013-X © 2013 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/B978-0-12-415783-5.00013-X

and co-medications of the patient, will dictate the mostappropriate specimen to collect, at the most appropri-ate time. In addition, specimen collection using thewrong anticoagulant and environmental factors suchas heat and light can affect drug stability and alter theconcentration of therapeutic drugs. TDM measure-ments are typically collected after a patient is pre-dicted to have achieved steady-state concentrationfrom predose (trough) collections, which occur beforethe next scheduled dose. Steady state is achieved whenthe concentration of drug in the body is in equilib-rium with the rate of dose administered and the rateof elimination (after initiation of therapy, the timeequivalent to at least five half-lives of the drug isneeded for establishment of steady state). A randomcollection may reflect steady state for some drugs,particularly those with long elimination half-lives.Random collections are also important when evaluat-ing therapeutic failure, toxicity, or overdose situation.Consideration of inaccuracies and appropriate inter-pretation of results depends on understanding thepre-analytical circumstances surrounding a requestfor TDM.

Specimen collection containers can alter TDMresults by affecting drug concentration and propor-tional stability. Failure to separate cells promptly fromserum or plasma may lead to in vitro metabolism, ashas been observed with fosphenytoin and mycopheno-lic acid acyl glucuronide [2,3]. Serum versus plasma,variation among tube preservatives, and gel separatortubes are relevant for accurate recovery of certain

drugs or fractions of drugs. For example, heparin col-lection tubes can increase the concentration of free (notbound to proteins) drug concentration by activatinglipoprotein lipase and fatty acid concentration, whichwill displace protein-bound drug from albumin and α1

glycoprotein [4]. Free fraction of phenobarbital, phe-nytoin, and valproic acid was shown to be elevated inserum versus plasma, whereas the free fraction of car-bamazepine and theophylline was lower in serum ver-sus plasma [5]. The proportion of free valproic acidwas found to be time-sensitive, and it decreased signif-icantly after 96 hr of storage at ambient temperature,most likely due to degradation of binding proteins.Blood specimens that are exposed to extreme tempera-tures can lead to plasma protein degradation as welland increase the free fraction of drugs that are highlybound to protein (e.g., phenytoin and valproic acid).Inappropriate dose adjustments can be made whenbased on free drug concentrations determined withsuboptimal specimens. In addition, citrate collectiontubes were associated with a decrease in the total con-centration of valproic acid [6]. More dramatically, gelseparator tubes have the potential to decrease totaldrug recovery through adsorption of the drug to thegel material. Separator tubes were reported to decreasethe recovery of cardiac drugs, tricyclic antidepressants,anticonvulsants, and antipsychotics. As such, gel sepa-rator tubes should not be used as collection tubes forTDM unless validated to be appropriate [4].

Drug stability can also be influenced by sensitivityto light or heat and may lead to erroneous TDM

• Sample preparation• Assay design• Assay components• Detection prince

Pre-AnalyticalFactors

AnalyticalFactors

Interferences

• Best specimen• Timing of collection• Quality of specimen• Patient status• Co-medication

FIGURE 13.1 Factors that affect accuracy of aTDM result.

196 13. ISSUES OF INTERFERENCES IN THERAPEUTIC DRUG MONITORING

ACCURATE RESULTS IN THE CLINICAL LABORATORY

results if specimens are stored under inappropriateconditions. Amiodarone, methotrexate, chlordiazepox-ide, carbamazepine, chlorpromazine, haloperidol, andfluoxetine are examples of light-sensitive drugs. Rapidin vitro degradation is also recognized for bupropion,busulfan, carbamazepine, lithium, and olanzapine. Theconsequences of in vitro degradation include falselylow results, which could potentially contribute to inap-propriate dose adjustments. Specimens containingunstable drugs should be stored at the appropriatetemperature (i.e., frozen) to preserve integrity and pre-vent falsely low results. In addition, the impact ofrepeat freeze�thaw cycles on drug integrity should becarefully evaluated [4].

TDM results can also be affected by a patient’s clini-cal status and lifestyle factors such as diet, smoking,alcohol use, co-morbidities, pharmacogenetics, andpolydrug therapy. Food and fluid intake can alter thepharmacokinetics (absorption, distribution, metabo-lism, and elimination) of a drug by impacting gastricpH and emptying time, which can affect drug absorp-tion. Drugs that are administered orally are absorbedinto the bloodstream through the gastrointestinal tractby passive diffusion if the drugs are lipid soluble ornon-ionized. Factors that affect intestinal motility,hepatic blood flow, and bile flow will also have animpact on the pharmacokinetics of a drug. For exam-ple, protein intake can affect drug�protein binding fordrugs and alter drug clearance. In 1987, Fagan et al.demonstrated that high-protein diets can increase theclearance of propranolol and theophylline [7].

Food�drug interactions will influence drug bio-availability and drug metabolism by inhibiting orinducing drug-metabolizing enzymes. For example,grapefruit juice can inhibit the cytochrome P450 iso-zyme 3A4 (CYP3A4) in the small intestine and increasebioavailability of several drugs [8]. This isozyme, partof the superfamily of CYP enzymes associated withdrug metabolism, is involved in metabolism of approx-imately half of all drugs. Increased drug bioavailabilitycan lead to enhanced drug activity and possible toxic-ity. Drug classes that are affected by this food�druginteraction include calcium channel blockers (e.g.,nifedipine), antiarrhythmic drugs (e.g., amiodarone),benzodiazepines (e.g., diazepam), antiepileptic drugs(e.g., carbamazepine), antibiotics (e.g., erythromycin),antiretrovirals (e.g., indinavir), immunosuppre-ssants (e.g., cyclosporine A), and cholesterol-loweringdrugs (e.g., simvastatin). In addition, cruciferousvegetables such as cabbage and cauliflower and char-broil foods can induce metabolism of drugs metabo-lized by the CYP1A2 isozyme. Substrates of CYP1A2include theophylline, clozapine, and olanzapine.Alcohol consumption can also induce the CYP2E1isozyme to increase metabolism of certain drugs, in

addition to the well-recognized synergistic effect withthe pharmacodynamics of depressant drugs. Cigarettesmokers tend to require high doses of many therapeu-tic drugs to obtain optimal therapy, in part due to thefact that nicotine induces isozymes CYP1A1, CYP1A2,and CYP2E1.

Genetic polymorphisms in the genes that code fordrug-metabolizing enzymes can also affect an indivi-dual’s ability to biotransform drugs for activation orelimination (pharmacogenetics). For example, geneticpolymorphisms can cause a patient to be a poor orslow drug metabolizer. However, the overall impact ofinduction or inhibition of drug-metabolizing enzymes,whether due to interacting substances or genetic pre-dispositions, depends on whether the drug substrate isactivated or inactivated by the affected isozyme(s). Fordrugs that are inactivated by metabolism (e.g., tricyclicantidepressants and warfarin), poor metabolizers areat risk of accumulating drug and are at risk of drug-induced toxicity if standard dosing is administered.Rapid metabolizers are at risk of therapeutic failuredue to suboptimal dosing and will require higherdoses to achieve optimal therapy. The opposite is truefor drugs that are activated by metabolism (e.g., clopi-dogrel and codeine). Accurate TDM is an importanttool for optimizing dosing under conditions ofunpredictable pharmacokinetics, such as pharmacoge-netic variants and food�drug and drug�druginteractions.

Another source of unpredictable pharmacokineticsthat requires TDM to optimize dosing is patients whoare critically ill. Of particular concern are patients withimpaired renal, hepatic, gastrointestinal, or cardiovas-cular function. Renal disease will reduce the glomeru-lar filtration rate and reduce the clearance of drugsthat are eliminated via the kidney. Renal disease willalso impact drug�protein binding in patients with ure-mia, due to uremia toxins competing for drug-bindingsites to albumin, and increase the concentration ofnon-protein-bound (free) drug and therapeutic effect.Liver toxicity or disease will decrease production ofalbumin and other proteins that bind to drugs.Hypoalbuminemia will increase the fraction ofpharmacologically active drugs and may increase therisk of toxicity. Decreased liver function will reducefirst-pass metabolism and also impact expression ofCYP450 isozymes in general. Gastrointestinal diseaseor a history of bariatric surgery, malabsorptive disor-ders, or intestinal disease may impact absorption ofdrugs [9]. Cardiovascular disease will decrease cardiacoutput, tissue perfusion, drug disposition, and absorp-tion. Reduced blood flow to the liver will decreasedrug metabolism.

TDM is also necessary in pregnant or nursingwomen to accommodate changes in maternal

197SOURCES OF PRE-ANALYTICAL INTERFERENCES IN TDM


physiology and to protect the fetus or infant from tox-icity. Pregnant women have increased body fat, totalbody water, and plasma volume, which will decreaseplasma protein concentration and drug�protein bind-ing. Lipophilic drugs will distribute and accumulate infat and decrease bioavailability. Drugs that are hydro-philic will have a lower volume of distribution in thebody and may have increased clearance and decreasedbioavailability in patients with excessive body fatcontent. Cardiac output in pregnant women isincreased, resulting in enhanced drug metabolism. Ageneral recommendation is to carefully monitorpregnant or nursing women who require drug therapyto ensure efficacy and safety for both mother and herunborn child [10].

SOURCES OF ANALYTICALINTERFERENCES IN TDM

The vulnerability of an analytical method to inter-ferences is dependent on the sample matrix, the sam-ple preparation method, the assay design, assaycomponents, and detection principle (see Figure 13.1).Sources of analytical interferences may be endogenousor exogenous. Laboratories must consider commonsources of interferences, such as hemolysis, duringdevelopment and validation of analytical methods,and they must investigate suspected interferences thatarise with analytical methods in routine use.Unfortunately, analytical interferences may go unrec-ognized by laboratories unless TDM results are ques-tioned by clinicians who have carefully evaluatedresults in the context of a specific patient scenario.Examples of analytical interference sources affectingcommon technologies used to support TDM are listedin Table 13.1.

Endogenous interferences (matrix components)known to affect TDM include classical biological inter-ferences such as hemolysis, bilirubinemia, hematocrit,and lipemia. For example, hemolysis is known tocompromise lithium quantitation due to a dilutioneffect. Both hemolysis and bilirubinemia may interferewith spectral detection methods, particularly for auto-mated homogeneous immunoassays [11]. Also, thetacrolimus II MEIA (microparticle enzyme immunoas-say) has been shown to be affected by hematocrit,wherein a tacrolimus result is biased if the bloodsample hematocrit falls outside 30�40% [12].Electrolytes and serum protein affect ion-transfervoltammetry methods, such as in quantification ofpropranolol, or ion-selective electrodes used for lith-ium quantitation [13]. Heterophilic antibodies, ster-oids, and other endogenous substances provide

interesting case reports for isolated, patient-specificinterferences with certain technologies. Examples areprovided later in this chapter. It can be stated thatbiological samples are very complex and essentiallyall endogenous compounds have the potential tointroduce inaccuracies through interferences, depend-ing on the unique characteristics of an individualpatient specimen, coupled with the analytical methodemployed.

Exogenous interferences are introduced from out-side the patient and add another level of sometimesunpredictable complexity. Such interferences may bequalified as direct or indirect. A direct exogenousinterference is related to the drug of interest, includingdrug metabolites, or drug impurities. Direct interfer-ences can and should be carefully evaluated duringassay development and validation. Compounds thatinterfere with a TDM technology but are unrelated tothe specific drug of interest could be qualified as indi-rect exogenous interferences and may be less intuitive.

TABLE 13.1 Example Sites and Sources of AnalyticalInterferences

AnalyticalMethod

Major Vulnerabilities toInterference

Example Sourcesof Interference

Immunoassay Antibodies (species, clone,epitopes)

Drug metabolites

Assay design (homogeneousvs. heterogeneous, specificsteps, timing of reactions,reagents, detection)

Similar drugs insame drug class

Any structurallysimilar compoundsHeterophilicantibodiesMatrix components

Chromatography Sample preparation(extraction, dilution, proteinprecipitation)

Any co-elutingcompoundsCompounds withsimilar detectioncharacteristicsMatrixcomponentsCarryover

Assay design (chemistry ofphases, flow rates,temperature)Detection technology

Massspectrometry

Sample preparation Isobaric or cross-talking compounds

Assay design (SIM, MRM,ions detected)

Impurities inreagents

Instrument (mass resolution,ionization, voltages, datacollection parameters,signal/noise)

Matrix components

Carryover

MRM, multiple reaction monitoring; SIM, selected ion monitoring.



Examples of indirect interferences include co-medica-tions, herbal medicines, vitamin and mineralsupplements, social drugs (e.g., nicotine and ethanol),components of the diet, or components of the patient’senvironment (e.g., water and air sources). Indirectinterferences may be predicted and characterized dur-ing assay development and validation, but they aremore often recognized and described after an assay isin routine use.

Analytical methods should be designed to detect,minimize, or compensate for predictable interferences.Some interfering substances may be minimizedthrough sample preparation methods such as dilution,protein precipitation, liquid�liquid or solid-phaseextraction. Physical separation techniques (e.g., chro-matography, ultrafiltration, and ultracentrifugation)may also help minimize matrix effects. Most auto-mated immunoassays do not incorporate any samplepreparation methods and are consequently at higherrisk from interferences. However, if the source of apotential interference is defined and is measureable(e.g., hematocrit), patient specimens could be qualifiedfor testing in advance. Samples that exceed establishedthresholds for a known interference could be disquali-fied for testing. To avoid inaccuracies in TDM results,alternate methods could be made available fordisqualified specimens.

All TDM results should be interpreted within thecontext of the clinical, pre-analytical, and analytical

scenario. Any result that is inconsistent with expecta-tions should be investigated. Figure 13.2 illustrates apossible algorithm for investigating a discordant TDMresult, with intent to guide identification or characteri-zation of the interference. Thus, an investigationshould begin by considering pre-analytical and analyti-cal variables. If suspicions surround the pre-analyticalcomponents of testing, a new specimen should be col-lected. If suspicions are aligned better with analyticalcomponents of testing, an alternate method should besought. Repeat testing of the original sample with theoriginal technology is also an important preliminarystep for investigating the possibility of an analyticalinterference. Comparing results between technicallydistinct analytical approaches is very informative.Additional tools that may help characterize or resolveinterference include dilution studies, to potentiallyreduce the impact of an interference sufficiently torestore accuracy of results; removing plasma compo-nents and washing red cells for assays based on detec-tion of erythrocyte-bound drug; and ultrafiltration orprotein precipitation for physically removing large-sized or protein-based interfering substances or sampleextraction methods for lipid-based interfering sub-stances. Solid-phase extraction, liquid�liquid extrac-tion, or other sample cleanup steps should be alignedwith the specimen type and the drug(s) of interest[14,15]. Many other approaches to characterizing, elim-inating, and preventing interferences may be

DiscordantTDM result

Analyticalfactor

Pre-analyticalfactor

Evaluatesample timing

and quality

Repeattesting

Characterizeremove

interference

Proteinprecipitation

Dilutionstudy

Sampleextraction/cleanup

Analyze byalternatemethod

Collect newsample

Evaluatepatient PK

Ultrafiltration

FIGURE 13.2 Algorithm for evalu-ating a discordant TDM result.

199SOURCES OF ANALYTICAL INTERFERENCES IN TDM


appropriate based on the actual source of interferenceand the available analytical technology.

MECHANISMS OF ANALYTICALINTERFERENCES IN TDM

Most well-described analytical interferences thataffect TDM are associated with commercially availableimmunoassays. Among immunoassay technologies,the source of the interference may relate to theantibodies on which the test is built or to the assaydesign. Immunoassays are available in homogeneousand heterogeneous formats (see Chapter 6), whichhave unique vulnerabilities to interferences based ontiming or various assay steps (e.g., washes and incuba-tions), chemistry (e.g., pH), temperatures, reactions(e.g., read times), and detection technology (e.g., spec-tral). All immunoassay formats employ a captureantibody and are typically designed to detect a singlerepresentative chemical structure against which thecapture antibody is raised. Sometimes, immunoassaysemploy more than one antibody, and the antibodiesmay have originated from several different species(e.g., mouse and rabbit). Actual performance character-istics and vulnerabilities to specific sources of analyti-cal interferences vary tremendously amongcommercial products, based in part on the antibodiesthat are unique to each product. In general, immunoas-says that utilize polyclonal antibodies are morelikely to be associated with analytical interferencesthan are those that utilize monoclonal antibodies dueto the fact that polyclonal antibodies have a largernumber of epitopes available for binding drug anti-gens with similar chemical structures. Also, in general,heterogeneous assay formats are less vulnerable tononspecific interferences than are homogeneous assayformats.

For drug classes that contain many structurally sim-ilar compounds, cross-reactivity across the drug classinherently compromises specificity for an individualdrug [16]. For example, immunoassays designed todetect tricyclic antidepressants generally detect severalcommon tricyclic antidepressant drugs at similar con-centrations. Because an acute intoxication with a tricy-clic antidepressant is likely to be managed based onthe drug class and clinical presentation, rather thanbased on the individual drug(s) responsible, analyticalinterference across the class of drugs is desirable andcan be useful clinically for drug detection, such as inthe emergency department, or for a psychiatric clinicthat wants to evaluate compliance with therapy.However, an immunoassay for tricyclic antidepres-sants is generally not useful clinically for TDM due tothe fact that other tricyclic structures are known to

cross-react with common immunoassays. Such assaysare also not useful for supporting TDM or pharmacoki-netic evaluations or whenever concentrations of indi-vidual drugs and drug metabolites are clinicallyrelevant. In the case of tricyclic antidepressants, thepreferred technology for analysis is liquid chromatog-raphy or liquid chromatography coupled to tandemmass spectrometry (LC-MS/MS). In any case, reviewof the cross-reactivity profile for any immunoassayproduct is required to evaluate which drugs and othercompounds are likely to be detected and with whatdegree of sensitivity/specificity. See the discussion oftricyclic antidepressants and other specific drug exam-ples for more information.

Examples of specific immunoassay designs that areassociated with analytical interferences known toadversely affect TDM (exemplified later in this chap-ter) include fluorescent polarization immunoassay(FPIA), enzyme multiplied immunoassay (EMIT),microparticle enzyme immunoassay (MEIA), andchemiluminescence immunoassay (CLIA).

CHROMATOGRAPHY ANDMASS SPECTROMETRY

Chromatography has long been recognized to impartspecificity to TDM because of the inherent ability toseparate components of the matrix and, hence, to sepa-rate drugs and drug metabolites from one another. Assuch, chromatographic techniques physically separatethe sample and are frequently referred to as physicaltechniques. Chromatographic methods are typicallydeveloped by individual laboratories and vary tremen-dously relative to sample preparation methods, chemis-try of related solid (e.g., column) and mobile (e.g., gasor liquid) phases, flow rates, temperatures, instrumentplatforms and associated capabilities, detection technol-ogies, methods of data collection, and extent of dataanalysis. Chromatographic methods are extremelyvulnerable to interferences from co-eluting compoundsthat may or may not be structurally similar to the com-pounds of interest. The relevance of a co-elutingsubstance depends on whether that substance interfereswith the analyte signal that is generated, which islargely based on the signal-to-noise ratio, and on thedetection method. Common detection methods includespectral (e.g., ultraviolet and fluorescent), electrochemi-cal, and mass spectrometry. Despite possible limita-tions, chromatographic methods, particularly coupledwith mass spectrometric detection, are recognized toresolve many of the interferences that are encounteredwith immunoassay methods and are generally regardedas gold standard platforms for TDM.



Mass spectrometry is a highly selective detectiontechnology that detects mass-to-charge ratios of ion-ized molecules and fragments. This detection technol-ogy is frequently coupled to gas or liquidchromatography. Output of data can be quantitative,qualitative for targeted masses, compared to a libraryof data to identify a particular compound, or used todetermine element composition and structure. Assayscan be designed using selected ion monitoring, whichis a mode that scans a limited mass-to-charge range;multiple reaction monitoring, in which an ion mass isselected in the first mass spectrometer and the frag-ment ion mass is selected in the second mass spec-trometer; or full mass spectrum, which scans a widemass-to-charge ratio range.

Although mass spectrometric methods are very spe-cific, the technology is not exempt from interferences.Ion suppression or enhancement, which alters ionintensity/abundances and signal-to-noise ratios, havebecome well recognized in mass spectrometric meth-ods but may go unrecognized when isolated to a singlepatient specimen. For example, by affecting analyteionization, ion suppression can occur in the presenceof compounds that are less volatile than the analyte ofinterest. Salts, anions, ion-pairing agents (e.g., trifluoro-acetic acid), drugs/metabolites, uncharacterized samplematrix components and co-eluting compounds, andreagent impurities are known to produce ionsuppression or enhancement. In electrospray ionization,the primary source of ion suppression is due tochanges in spray droplet formation in the presence ofnonvolatile compounds [17,18]. Another source ofinterference in mass spectrometry that may be over-looked includes compounds that are isobaric or isomet-ric to the analyte of interest. An example of isobaricinterferences was observed with endogenous nucleo-sides and ribavirin in an LC-MS/MS method [19].Plasticizers, reagents, lipids, and phospholipids in par-ticular are well recognized to introduce matrix effects,manifested as either suppression or enhancement ofanalytical response [20]. Consequently, internal stan-dards, such as stable isotope-labeled compounds, areused to minimize the effects of ion suppression andimprove the analytical accuracy of mass spectrometry[21]. Lastly, cross talk can cause interference when dif-ferent parent ions fragment and have identical production masses [22].

EXAMPLES OF INTERFERENCESTHATAFFECT TDM

In this section, specific examples are given regard-ing interferences affecting the measurement of varioustherapeutic drugs.

Interferences in Digoxin Measurement

Digoxin (Lanoxin) is a cardiac glycoside with posi-tive inotropic effects that is used to treat cardiacarrhythmias and congestive heart failure. Effects aredose-related, and TDM is required because the clinicalsymptoms of inadequate and excessive dose are simi-lar. The traditional therapeutic range for digoxin,0.5�2.0 ng/mL, is narrow among drugs that requireTDM. It is also noteworthy that the upper limit of thetherapeutic range overlaps somewhat with the thresh-old for toxicity. In congestive heart failure, an evenmore narrow range has been proposed (0.5�0.8 ng/mL),and patient outcomes begin to decline at concentrationsgreater than 0.9 ng/mL. The clinical expectations formaintaining a very narrow circulating concentration ofdigoxin raise questions about whether analytical techni-ques available for TDM of digoxin can perform withadequate accuracy and precision [23]. A very narrowtherapeutic range also demands that pre-analytical vari-ables be recognized and managed.

Pre-Analytical Variables

Digoxin has a high volume of distribution andrequires several hours to distribute after dosing. Acommon cause of an unexpectedly high digoxin istherefore based on inappropriate timing of specimencollection. Blood for digoxin analysis should be col-lected at least 8 hr after a dose is administered.

Analytical Variables

Both positive and negative interferences have beendescribed for immunoassays designed for TDM ofdigoxin. It is usually assumed that negative interfer-ence is most dangerous to the patient managementbecause it may contribute to inappropriate doseincreases. Sources of analytical interferences that arerecognized include digoxin metabolites; endogenousdigoxin-like immunoreactive factor or substance (DLIFor DLIS); and exogenous drugs, including anti-digoxinFab fragments used to treat digoxin overdose, aldoste-rone antagonists, other cardiac glycosides, and severalherbal medicines. Some forms of interference can beeffectively removed through physical separation,accomplished by ultrafiltration or chromatography.Analysis of ultrafiltrate, chromatographically distinctfractions, or direct analysis of independent specimencomponents such as via mass spectrometric detection,further reduces the potential for analytical interfer-ences [24].

Tables 13.2 and 13.3 summarize examples ofinterferences described for common immunoassaytechnologies in the absence or presence of digoxin,respectively. The type of interference described (posi-tive, negative, or none) for FPIA, EMIT, MEIA-II, and

201EXAMPLES OF INTERFERENCES THAT AFFECT TDM


CLIA is indicated, along with whether or not ultrafil-tration will resolve the interference. The data presentedin these tables are intended for example purposes andare inherently simplified. For example, the tables donot reflect that the presence and magnitude of interfer-ences is closely related to the concentration of interfer-ing substances and/or digoxin, and that specificreagents that utilize these technologies may differ. Thepackage insert for a specific assay of interest should beconsulted, and independent validation studies shouldbe performed as needed, to fully characterize any inter-ference and its potential impact to a unique clinical set-ting. In the following sections, each major source ofinterferences is described in more detail.

DIGOXIN METABOLITES

Digoxin is extensively metabolized. Cross-reactivityof the metabolites with antibodies used to developdigoxin immunoassays was historically a source ofanalytical interference, such as with radioimmunoas-says. In particular, the hydrolysis metabolites digoxi-genin, digoxigenin monodigitoxoside, and digoxigeninbisdigitoxoside exhibited near equal cross-reactivitywith digoxin in some assays. Because these metabolitesexhibit physiological activity, it was proposed that thiscross-reactivity could represent a total “bioactive”digoxin concentration [25,26]. Newer assays exhibit littlecross-reactivity to metabolites, suggesting that currentassays are not subject to the same cross-reactivity. Theconcentration of active metabolites is also not thought tobe sufficiently high to contribute significantly to the effi-cacy or toxicity of digoxin in most patients. Nonetheless,awareness of cross-reactivity to digoxin metabolites maybe relevant for application of immunoassay testing topatients who exhibit poor elimination kinetics, who maybe at risk for accumulation of clinically relevant drugmetabolites. Because metabolites do accumulate in urine,the analytical method selected for testing urine fordigoxin should be evaluated carefully.

DIGOXIN-LIKE IMMUNOREACTIVE FACTORS

The chemical structure of digoxin is steroid-like andtherefore exhibits high potential for endogenous coun-terparts. Indeed, a source of endogenous interferencesin digoxin assays has been recognized in select popula-tions in the absence of digoxin exposure or administra-tion. This steroid-like substance was called DLIF andDLIS in the mid-1980s and was shown to cross-reactwith digoxin immunoassays. DLIF has been recog-nized most for patients with conditions of volumeexpansion. Vulnerable populations include infants andyoung children, pregnant women, patients withimpaired renal or hepatic function, and persons whohave exercised vigorously. It has also been reported tobe elevated immediately after a myocardial infarctionand in the critically ill [25].

It is now thought that there are at least two classesof DLIF that may or may not cross-react with digoxinimmunoassays and exert activity at endogenoussodium�potassium ATPase [27]. The degree and typeof interference may change in the presence of digoxin.In the absence of digoxin, clinically significant concen-trations of apparent digoxin (no actual digoxinpresent) have been observed in samples collected fromnewborns and pregnant women and analyzed by FPIA[28]. In the presence of digoxin, DLIF may lead topositive or negative interference, based on the specificanalytical technology. Ultrafiltration or chro-matographic separation remains the best means of

TABLE 13.2 Interferences with Digoxin Immunoassays in theAbsence of Digoxin

Interferent FPIA EMIT MEIA-II CLIA Resolved byUltrafiltration

DLIF 1 1 Yes

Drugs

Digitoxin 1 1 1 1 Partial

Anti-digoxinFab

1 1 1 Yes

Aldosteroneantagonists

1 1 1 1 No

Herbals

Chan su 1 1 1 Partial

Danshen 1 Yes

TABLE 13.3 Interferences with Digoxin Immunoassays in thePresence of Digoxin

Interferent FPIA EMIT MEIA-II CLIA Resolved by

Ultrafiltration

DLIF 1 1 - Yes

Drugs

Digitoxin 1 1/2 Partial

Anti-digoxinFab

1/2 1/2 1/2 Yes

Aldosteroneantagonists

1 1 2 1 Partial

Herbals

Chan su 1 1 2 Partial

Danshen 1 2 Yes

CLIA, chemiluminescence immunoassay; EMIT, enzyme multiplied

immunoassay; FPIA, fluorescent polarization immunoassay; MEIA,

microparticle enzyme immunoassay.



resolving this type of interference, when suspected.Due to the extensive characterization of DLIF andimprovements in immunoassay design, currently avail-able immunoassays may exhibit minimal or no clini-cally significant interference from DLIF [29,30].

ANTI-DIGOXIN IMMUNE FRAGMENTS

Digibind and DigiFab are examples of pharmaceuti-cal immune (Fab) products that bind digoxin withhigh affinity, and they are used as antidotes to treatdigoxin overdose. These Fab products are responsiblefor variable amounts of analytical interferencewith digoxin assays, both in the absence and in thepresence of digoxin. Technologies such as CLIA,MEIA, and EMIT demonstrate concentration-dependent positive interference in the absence ofdigoxin, and concentration-dependent negative inter-ference is observed in the presence of digoxin, to thepoint of molar equivalency (neutralization). As mightbe expected, the presence of digoxin under conditionsof Fab excess results in positive interference [31].

Patients undergoing Fab-based decontaminationtreatment can be monitored by measuring free digoxinin ultrafiltrate or by incorporating a pre-analyticalprotein-precipitation step to measure total digoxin(e.g., FPIA). Monitoring free digoxin concentrations inplasma ultrafiltrate is the most accurate and acceptedapproach. It is important to employ a method of analy-sis such as FPIA or MEIA that is free of matrix effectsbecause ultrafiltrate exhibits different chemical/physi-cal properties than plasma or serum. Ultrafiltrate is notan appropriate specimen for methods that depend onsample fluid dynamics, such as slide technologies (e.g.,Vitros) [32].

CARDIAC GLYCOSIDES

The digitalis family is composed of several cardiacglycosides with similar chemical structures, includingthe once popular drug digitoxin. Whereas it is not cur-rently used in the United States, digitoxin is used inEuropean, Scandinavian, and other countries.Although it would not be expected that digoxin wouldbe co-administered with digitoxin, a patient may betransitioned from one drug to another or may be usingundisclosed medication obtained outside the primaryclinic. The therapeutic dose and corresponding thera-peutic concentration range of digitoxin is significantlyhigher (10- to 15-fold) than that of digoxin. Due to thehigher therapeutic range, and structural similarity,even low cross-reactivity with a digoxin immunoassaycould produce clinically significant interference. Onlythe CLIA digoxin assay has sufficiently low cross-reactivity with digitoxin to avoid analytical interfer-ences. FPIA assays are particularly vulnerable topositive interference from digitoxin in the absence of

digoxin. MEIA assays are moderately susceptible topositive interference from digitoxin in the absence ofdigoxin. Of note, negative interference is observedwith MEIA when digoxin and digitoxin are present.Other cardiac glycosides, including oleandrin (used infolk medicines), may exhibit the same patterns of posi-tive interference in the absence of digoxin and eitherpositive or negative interference in the presence ofdigoxin. Interferences from nondigoxin cardiac glyco-sides may be at least partially resolved by measuringfree digoxin in ultrafiltrate [33,34].

ALDOSTERONE ANTAGONISTS

Spironolactone and potassium canrenoate are usedto treat hypertension and congestive heart failure, andthey are often co-prescribed with digoxin. These co-medications, along with their common active metabo-lite canrenone, are structurally similar to digoxin andhave been described to produce positive interferencein FPIA and CLIA digoxin assays. Negative interfer-ence has also been described for MEIA digoxin assays[35]. Newer-generation MEIA III assays that employmonoclonal antibodies have improved resistance to thenegative analytical interferences described. However,clinically significant positive analytical interference hasbeen described for the MEIA III assay in patientsco-medicated with higher-dose spironolactone (e.g.,100 mg/day). Similar positive interferences aredescribed with EMIT digoxin assays. The impact of theanalytical interferences on dosing can be significant,easily exceeding the therapeutic range of digoxin. Onestudy demonstrated clinically significant differences inapproximately 45% of patients co-medicated withdigoxin and either spironolactone or canrenoate. Suchinterferences were not eliminated by measurement ofultrafiltrate [36]. As such, digoxin monitoring forpatients co-medicated with aldosterone antagonists isbest pursued with chromatographic techniques.

HERBAL MEDICINES

Herbal medicines are a cornerstone of traditionalEastern medicine and lifestyle, and they are readilyavailable without prescription. These medicines arenot well controlled in terms of potency or purity, andthey may not be reported by patients who use them.Several case examples and in vitro studies havedemonstrated that select herbal medicines producebidirectional analytical interferences in digoxin immu-noassays. For example, positive and negative interfer-ences have been described extensively for Chan su andDanshen. Thus, Chan su produced concentration-dependent positive interference in FPIA, EMIT, andMEIA digoxin immunoassays in the absence ofdigoxin. In the presence of digoxin, Chan su producedpositive interference in FPIA and EMIT and negative



interference in MEIA. Chan su did not affect CLIAwith or without digoxin present. Interferences withChan su were only partially resolved by analysis ofultrafiltrate. Positive interference from Danshen hasbeen reported in the absence of digoxin when analyzedby FPIA but not by EMIT, MEIA, or CLIA. As withChan su, positive interference has been observed withFPIA and EMIT, and negative interference with MEIA,when Danshen is found in the presence of digoxin; noeffect was observed with CLIA. Danshen interferenceswere largely resolved by analysis of ultrafiltrate[37�39].

CASE REPORT1 A 19-year-old Taiwanese femalepresented to an emergency department with nausea,vomiting, dizziness, weakness, and numbness in themouth. Noteworthy was her admission to havingingested cooked toad eggs approximately 1 hr prior.Vitals and neurological exam were normal, but an elec-trocardiogram revealed a first-degree atrioventricularblock. Serum digoxin concentration was measured byimmunoassay. Despite no history of digoxin therapy,the concentration was 1.9 ng/mL; potassium concen-tration was 7.1 mmol/L. The patient was administeredanti-digoxin Fab and admitted to the intensive careunit. Both the apparent digoxin concentration andpotassium concentrations declined, and the patientwas released a few days later. The most likely cause ofthe digoxin cross-reactivity is that the toad eggsingested contained Chan su, a traditional Chinese aph-rodisiac commonly made from toad venom.

Interferences in Carbamazepine Measurement

Carbamazepine (Tegretol and Carbatrol) is a tricyclicanticonvulsant drug used for the treatment of partialand tonic�clonic seizures, trigeminal neuralgia, andmanic depression. The therapeutic range forcarbamazepine is 4�12 μg/mL; signs of toxicity, such asstupor, seizures, and respiratory depression, may occurat concentrations exceeding 15 μg/mL. Carbamazepineis metabolized primarily by cytochrome P450 isozyme(CYP) 3A4 to the active metabolite, carbamazepine-10,11-epoxide. The 10,11-epoxide metabolite has similaractivity as the parent drug, and it may contribute signifi-cantly to efficacy of the drug in populations thataccumulate the metabolite, such as children. The concen-tration of the metabolite may be higher than the parentdrug concentrations in situations of carbamazepineoverdose and in patients with renal failure (uremia);therefore, monitoring drug ratios of parent andmetabolite may be useful for evaluating compliance anddrug�drug interactions.


Drug�drug interactions are common with carba-mazepine due to relatively high protein binding(65�80%) and involvement of drug-metabolizingenzymes, including CYP2C19 and CYP3A4.Carbamazepine is eliminated primarily via the kid-neys; thus, renal failure or insufficiency can lead toelevated results and increase the risk for toxicity.


Major sources of analytical interference forcarbamazepine include cross-reactivity from the10,11-epoxide metabolite and structurally similardrugs. Cross-reactivity of carbamazepine and itsmetabolite can vary in commercial immunoassays,from 0% for the Vitros assay to approximately 93.6%on the Dade Dimension. Other assays, such as MEIA(AxSYM), have moderate cross-reactivity (22%) [41].This cross-reactivity can be utilized to calculate thetotal amount of epoxide present, but chromatographicmethods are preferred [42]. Analytical interferenceswith carbamazepine immunoassays have beendescribed for drugs that are structurally similar tocarbamazepine, such as oxcarbazepine, which producepositive interference in carbamazepine analysis byEMIT [43]. Cross-reactivity with carbamazepineanalogs that are currently in development, such as esli-carbazepine, is not yet characterized. Because this newdrug is a purified isomer, it is important to recognizethat nonchiral chromatographic assays would not beable to distinguish between use of eslicarbazepine anduse of racemic licarbazepine. Noncarbamazepineanalogs may also cross-react with carbamazepineassays. For example, Parant et al. reported that theantihistamine drug hydroxyzine and its metabolite,cetirizine, produced a false-positive result for carba-mazepine using the particle-enhanced turbidimetricinhibition immunoassay (PETINIA) but not with EMITor turbidimetry (ADVIA Centaur) [44,45].

Interferences in Phenytoin Measurement

Phenytoin (Dilantin) is administered to manage gener-alized tonic�clonic seizures, partial or complex�partialseizures, and status epilepticus. There is a goodcorrelation between plasma concentration of phenytoinand its clinical effect, and a well-accepted therapeuticrange is 10�20 μg/mL. Fosphenytoin (Cerebyx) is a pro-drug that is rapidly converted by hydrolysis to phenyt-oin. Total plasma phenytoin concentrations greater than20 μg/mL do not enhance seizure control and are associ-ated with adverse effects such as nystagmus, ataxia,psychosis, hyperglycemia, nystagmus, and hematological1From Kuo et al. [40].



disorders; concentrations greater than 35 μg/mL havebeen shown to precipitate seizure activity.


Drug�drug interactions are common with phenyt-oin due to high protein binding (. 90%) and involve-ment of drug-metabolizing enzymes, includingCYP2C9 and CYP2C19. Phenytoin is primarily elimi-nated via the kidneys; thus, renal failure or insuffi-ciency can lead to elevated results and increase the riskfor toxicity due to accumulation of the parent drug.Samples collected after administration of fosphenytoinmay lead to falsely elevated results due to in vitrometabolism of fosphenytoin, particularly in patientswith elevated alkaline phosphatase activity [2].


A major source of analytical interference forphenytoin is the primary metabolite 5-(p-hydroxyphe-nyl)-5-phenylhydantoin (HPPH). Several phenytoinimmunoassays cross-react with the metabolite HPPH,leading to falsely elevated results [46,47]. The glucuro-nide form of HPPH is eliminated in urine. Analyticalinterference due to HPPH is of particular concern foruremic patients as well as any patients who have poorrenal function and may thereby accumulate the metab-olite [48].

Other sources of analytical interference reported forphenytoin include the nonsteroidal anti-inflammatorydrug oxaprozin (Daypro), which can exhibit cross-reactivity with TDx phenytoin assays to produce afalsely elevated result [49,50]. Fosphenytoin cross-reacts directly with several immunoassay formats,including FPIA, EMIT, and CLIA, to produce falselyelevated results. Of interest, the extent of cross-reactivity increases in the presence of phenytoin forFPIA (TDx) but is independent of phenytoin concen-tration for CLIA (ACS:180) [51]. False-negative resultsare also possible, as demonstrated by Brauchli et al. ina patient with monoclonal IgM-λ and testing per-formed by an automated particle-enhanced turbidimet-ric inhibition immunoassay (see case report) [52].

CASE REPORT2 A 73-year-old female was admittedto a hospital due to confusion and recurrent convul-sive facial contractions. The convulsions increased andextended to the right side of her body. An EEG identi-fied epileptic zones in the occipital lobe, and treatmentby phenytoin infusion was initiated. Despite consistentdaily dosing, total serum phenytoin concentration wasundetectable (,0.4 ng/mL) by PETINIA (DimensionAnalyzer, Siemens Diagnostics) in multiple samples.Other medications administered to the patient were

detected in the same samples, eliminating the possibil-ity of sample mix-up. The possibility of drug�druginteractions was also eliminated. Because of the unex-pected result, testing by alternate methods was pur-sued. Phenytoin was detected and concentrations werereasonable when analysis was performed by FPIA orby chromatographic methods. Testing with PETINIAwas repeated after a protein precipitation step, andphenytoin was detected as expected. It was thereforehypothesized that the false-negative results were dueto an interfering protein. A monoclonal IgM-λ wasdetected in the patient serum by immunofixation, andthis was likely responsible for the analyticalinterference.

Interferences in Measurement ofImmunosuppressant Drugs

Immunosuppressant drugs (cyclosporine A, tacroli-mus, sirolimus, everolimus, and mycophenolate mofetil)function to suppress the immune system by inhibitingT cell activation and proliferation. Immunosuppressantdrugs are used to treat autoimmune disease, allergies,multiple myeloma, and chronic nephritis and to preventrejection after organ transplantation. TDM for immuno-suppressant drugs is routinely performed in thetransplant setting, and dose adjustments are madebased on results. Blood concentrations below thetherapeutic range can lead to underdosing, which canput a patient at risk for graft rejection because theimmune system is not suppressed. Blood concentra-tions that exceed the therapeutic range can lead tooversuppression of the immune system and increasethe risk of opportunistic infections, malignancy, andorgan toxicity.

Cyclosporine (Sandimmune and Neoral) and tacroli-mus (Prograf) are both calcineurin inhibitors and haveserious adverse effects, such as renal dysfunction andtoxicity, hypertension, and hyperlipidemia, with excessexposure. The general therapeutic range is100�400 ng/mL. The therapeutic range for tacrolimusis 5�20 ng/mL. Sirolimus (Rapamune) and everolimus(Zortress and Afinitor) are inhibitors of the mamma-lian target of rapamycin (mTOR) and function toinhibit T-lymphocyte activation and proliferation. Theyare also associated with serious adverse effects, suchas anemia, leucopenia, thrombocytopenia, hyperlipid-emia, and gastrointestinal effects. The therapeutic rangefor sirolimus is 12�20 ng/mL, and it is 3�8 ng/mLfor everolimus. Mycophenolate mofetil (CellCept) is aprodrug that is rapidly metabolized to mycophenolicacid (MPA) in the liver. MPA is a reversible andnoncompetitive inhibitor of inosine monophosphatedehydrogenase, which is important for guanine2From Brauchli et al. [52].



nucleotide synthesis for T cell proliferation. MPA is asso-ciated with adverse effects such as leukopenia, diarrhea,and vomiting. The therapeutic range for predose MPA is1.0�3.5 mg/L when combined with cyclosporine.


Cyclosporine, tacrolimus, sirolimus, and everolimusare highly bound to red blood cells and proteins; there-fore, TDM is best performed with whole blood. MPAis not bound to red blood cells but highly bound toplasma proteins; therefore, TDM is performed withplasma. Changes in hematocrit may affect accuracy ofwhole blood testing results.

Bioavailability is extremely variable with most immu-nosuppressant drugs. These drugs are extensivelymetabolized and eliminated primarily in the urine.As such, patients with impaired gastrointestinal,hepatic, or renal function are subject tounpredictable pharmacokinetics. For example, livertransplant patients with hyperbilirubinemia willaccumulate tacrolimus metabolites leading to a high pos-itive bias in tacrolimus results by immunoassay [11].Drug�drug interactions that lead to elevated or reducedconcentrations of immunosuppressant drugs are also asignificant concern.


Immunosuppressant drugs can be monitored byimmunoassays and chromatographic methods (high-performance liquid chromatography (HPLC) andLC-MS/MS). LC-MS/MS methods are now commonlyused to create multianalyte panels and to increasespecificity compared to immunoassay methods [53].These methods are not free of interferences, and theyshould be evaluated with the same scrutiny as animmunoassay or other analytical method [20].Nonetheless, immunoassays remain a convenientmeans of testing, particularly in a hospital setting.Sources of analytical interferences for immunoassaysinclude drug metabolites and endogenous factors suchas heterophilic antibodies and hematocrit. In addition,the structural similarity between sirolimus and everoli-mus leads to near equivalent cross-reactivity with siro-limus immunoassays. Everolimus can be monitored bysirolimus immunoassay, but if a patient isco-medicated with both drugs, distinguishing everoli-mus and sirolimus requires a chromatographicapproach [54�56]. Functional assays designed tomonitor and optimize immunosuppressant therapy areavailable as well, particularly for MPA, but are notdiscussed here [57,58]. See Table 13.4 for a summary ofdrugs and drug metabolites that are reported to interferewith immunoassays for immunosuppressant drugs.

DRUG METABOLITES

Cyclosporine has 31 metabolites; one of the majormetabolites has approximately 10% of the immunosup-pressive activity of the parent compound, whereasthe rest of the metabolites are inactive. Tacrolimus

TABLE 13.4 Drugs and Drug Metabolites That May Interferewith Immunoassays for Immunosuppressant Drugs

Drug Metabolites Drugs

Cyclosporine(Sandimmune)

31 (inactive) Amikacin

Hydroxylatedcyclosporine (10%activity)

Amphotericin B

Azathioprine

Carbamazepine

Chloramphenicol

Cimetidine

Digoxin

Disopyramide

Erythromycin

Furosemide

Gentamicin

Kanamycin A

Lidocaine

Mycophenolicacid

Phenobarbital

Phenytoin

Prazosin

Prednisone

Tacrolimus (Prograf) 8 (inactive) Quinidine31-O-desmethyltacrolimus (active)

Rifampin

Sirolimus (Rapamune) Hydroxy-sirolimus(unknown activity)

Tobramycin

Desmethyl sirolimus(unknown activity)

Verapamil

Sirolimus (ring-openform; unknown activity)

Everolimus (Zortress,Affinitor)

Hydroxy-everolimus(unknown activity)Dihydroxy-everolimus(unknown activity)Desmethyl everolimus(unknown activity)Everolimus (ring-openform; unknown activity)

Mycophenolate mofetil(CellCept, Myfortic)

7-O-glucuronide(MPAG) (inactive)Acyl-glucuronide(AcMPAG) (active)



has 1 active metabolite, 31-O-desmethyl tacrolimus,and 8 metabolites that are inactive. Sirolimus has 7inactive metabolites, and everolimus has at least 20inactive metabolites. The primary metabolite of MPAis MPA-glucuronide (MPAG), which is pharmacologi-cally inactive.

Several immunoassay methods are known to cross-react with inactive metabolites to produce results thatare 20�60% higher than those obtained by chro-matographic techniques such as HPLC or LC-MS/MS[59�61]. Immunoassay methodologies that utilizepolyclonal antibodies exhibit the highest degree of pos-itive bias compared to immunoassays based on mono-clonal antibodies; however, LC-MS/MS is thepreferred technology. For MPA, immunoassay resultsmay be overestimated by cross-reactivity with theMPAG metabolite, as well as the prodrug mycopheno-late mofetil [62,63].

ENDOGENOUS INTERFERENTS

Heterophilic antibodies may interfere with immunoas-says of any sort, and they can be identified with variousdilution and pretreatment methods. Falsely elevatedtacrolimus and cyclosporine results were reported inpatients receiving these drugs (see case report), mostlikely due to endogenous anti-β-galactosidase antibo-dies [64]. Hematocrit values have been shown to corre-late with response in an MEIA assay for tacrolimus aswell. Specifically, low hematocrit values are associatedwith overestimated tacrolimus concentrations by MEIA;the overestimation can be accounted for by correcting forhematocrit [12].

CASE REPORT3 A 53-year-old male who received akidney transplant was treated with tacrolimus andcorticosteroids. Tacrolimus was monitored usingthe antibody conjugated magnetic immunoassay(ACMIA)-tacrolimus (Siemens Dimension). The tacroli-mus results obtained for the renal transplant patientwere consistent with expectations for the first 3 weekspost-transplant (, 12 ng/mL). An unexpected tacroli-mus result of 21.5 ng/mL was then observed, anddosing was suspended for a few days. During theperiod in which tacrolimus was discontinued, bloodtacrolimus concentrations persisted at greater than10 ng/mL. When tacrolimus was reintroduced, it wasmeasured by MEIA-tacrolimus (IMx), and results wereconsistent with clinical expectations. For comparison,samples were analyzed in parallel with ACMIA (onDimension analyzer: Siemens Diagnostics) and resultswere approximately twice the MEIA concentrations. Inthe automated ACMIA-tacrolimus method(Dimension), whole blood is lysed and mixed with

anti-tacrolimus�β-galactosidase antibody conjugateand magnetic particles to which unlabeled tacrolimusis bound. Tacrolimus in the patient blood competeswith the tacrolimus bound to particles for bindingwith the conjugate. The magnetic particles are sepa-rated from the reaction mixture, and the tacrolimus-bound conjugate is detected in the presence of enzymesubstrate. In the case report, a dilution study was per-formed that showed nonlinearity. In addition, theinterference was resolved completely by treatmentwith polyethylene glycol and by testing washed ery-throcytes, suggesting that the interferent was a plasmacomponent. Positive interference was observed withACMIA-cyclosporine as well, despite the fact that thepatient never received cyclosporine, which further sug-gests that the cause of the interference was endoge-nous anti-β-galactosidase antibodies.

Interferences in Measurement of Antidepressantand Mood-Stabilizer Drugs

Tricyclic Antidepressants

Tricyclic antidepressants (TCAs), such as amitripty-line (Elavil), clomipramine (Anafranil), doxepin(Sinequan, Prudoxin, Zonalon, and Silenor), imipra-mine (Tofranil), trimipramine (Surmontil), nortripty-line (Pamelor), and desipramine (Norpramin), arenamed for their three-ring structure and used to treatvarious forms of depression, anxiety disorders, eatingdisorders, attention deficit hyperactivity disorder,enuresis in children, and chronic and neuropathicpain. Plasma concentrations of TCAs have a positivecorrelation with clinical improvement and toxicity.Serum TCA concentrations greater than 500 ng/mLcan produce adverse anticholinergic symptoms,including dry mouth, sedation, blurred vision, fever,urinary retention, agitation, confusion, and seizures.TCA toxicity from overdose can be life-threateningand involve cardiovascular complications such ashypotension, tachycardia, and cardiac arrhythmias.Serum concentrations greater than 1000 ng/mL can befatal. Chronically administered TCAs can contribute tocardiac toxicity by accumulating in cardiac tissues[13,65].


Several TCAs have active metabolites that may beimportant to identify and quantitate because their con-centrations contribute to the therapeutic efficacy ofTCAs. Some active metabolites are available as inde-pendent drugs. For example, nortriptyline is the activemetabolite for amitriptyline, and desipramine is theactive metabolite for imipramine. Drug�drug ordrug�herb interactions involving cytochrome P4503From D’Alessandro et al. [64].



isozymes, such as CYP2D6 and CYP2C19, commonlycontribute to accumulation of active drug or drugmetabolites that may lead to concentration-relatedtoxicity [13,66].


Immunoassays designed to detect TCAs are proneto interferences from other TCA drugs and drug meta-bolites, as well as structurally similar compounds fromdifferent drug classes. Table 13.5 provides a list ofdrugs that have been reported to interfere with immu-noassays designed to detect TCAs. For example, cyclo-benzaprine, a skeletal muscle relaxant, has a similarstructure to TCA and can cause a false-positive resultfor TCA by immunoassay due to antibody cross-reactivity [67]. Phenothiazine antipsychotic drugs, suchas thioridazine, have three-ring structures and can alsocross-react with TCA antibodies and produce false-positive results. Other three-ring structured com-pounds can produce false-positive TCA results viacross-reactivity with TCA immunoassays, includingphenothiazine antipsychotic drugs such as thioridazine[68]; antiepileptic drugs such as carbamazepine andoxcarbazepine [69]; antihistamines such as cetirizine,hydroxyzine, and diphenhydramine [70]; and the anti-psychotic drug quetiapine [71].

HPLC methods are well-established for TCA testing;however, this methodology is prone to interferences inpeak separation and retention time, which can occur inpatients on multidrug therapy. Thioridazine, an anti-psychotic drug for schizophrenia therapy, can interferewith imipramine metabolism and analysis by HPLC[68]. Mass spectrometric methods provide high speci-ficity and minimize interferences; thus, they areconsidered the method of choice for identification,differentiation, and quantitation of TCA drugs [72].

CASE REPORT4 A 17-year-old female with a historyof bulimia was admitted to an emergency departmentafter unknown drug ingestion. The patient was drowsywith dilated, reactive pupils, slurred speech, andataxia. Electrolytes and liver function test results werenormal. Urine toxicology testing was negative, but aserum toxicology screen was positive for TCAs byFPIA. An electrocardiograph was pursued thatrevealed only sinus tachycardia. Targeted chro-matographic testing failed to identify any specific TCA,and the patient’s condition improved rapidly. Subsequenttesting revealed carbamazepine at 18.6 μg/mL in a bloodsample collected approximately 12 hr postingestion,which decreased to 10 μg/mL in a sample collected atapproximately 18 hr postingestion, but no TCAs,

demonstrating that the initial screening result was a falsepositive.

Lithium

Lithium carbonate (Eskalith and Lithane) is a mono-valent cation that is used as a mood-stabilizing agentfor treatment of bipolar disorder, acute manic episode,and depression. Lithium TDM is recommendeddue to its narrow therapeutic range (0.6�1.2 mmol/L),unpredictable serum concentrations, and concentration-dependent toxicity. Concentrations of lithium greaterthan 1.5 mmol/L are associated with lethargy, muscleweakness, tremors, and speech difficulties. Lithium con-centrations greater than 2.5 mmol/L can producemuscle rigidity, mental confusion, seizures, coma,cardiac arrhythmias, and death [13].


Patient specimens should not be collected in lithiumheparin tubes because the additive can increase theconcentration of lithium in patient samples, causing afalsely elevated result. For this reason, serum is pre-ferred over plasma. In addition, specimens that aregrossly hemolyzed may cause a dilution effect on lithiumconcentrations and should not be used for lithiumanalysis. Co-administration of diuretics and nonsteroidalanti-inflammatory agents can cause drug�drug interac-tions and affect lithium concentrations in serum by alter-ing lithium excretion. Lithium is excreted primarily inurine, where it is actively absorbed by the kidneys;hence, serum concentrations of lithium are affected bythe glomerular filtration rate [13].


Lithium is most frequently measured by ion-selective electrodes (ISE). Automated colorimetric,photometric, and enzymatic methods, as well aselemental analysis using flame atomic absorptionspectrometry, flame atomic emission spectrophotome-try, or inductively coupled plasma mass spectrometrymethods, are also available and could be used to

TABLE 13.5 Drug Analytes That May Interfere with TricyclicAntidepressant Immunoassays

Carbamazepine Maprotiline

Carbamazepine-10,11-epoxide Oxcarbazepine

Cetirizine Perphenazine

Chlorpromazine Phencyclidine

Cyclobenzaprine Prochlorperazine

Fluphenazine Quetiapine fumarate

Hydroxyzine Thioridazine

4From Matos et al. [73].



resolve a suspected interference with ISE [74]. Sourcesof analytical interferences are based on the technology.For ISE, sources of interference include ions originat-ing from other drugs and elements. Drugs such asquinidine, procainamide, and its metabolite, N-acetyl-procainamide, can produce a positive interference withlithium results by ISE, particularly with the Beckmananalyzer. The interference from the three drugs isadditive. Quinidine can also produce a negative bias inlithium results by colorimetric methods, and valproicacid can produce a positive bias in lithium results bythe same method. A positive bias in lithium results isalso caused by drug interferences from the cardiacdrug lidocaine and the anticonvulsant drug carbamaz-epine by ISE. Calcium concentrations greater than8.9 mmol/L can cause a positive bias interference onISE methods and a negative bias interference by colori-metric methods. Potassium and sodium can alsoproduce a negative and positive bias interference,respectively, by colorimetry [75].

CONCLUSIONS

TDM is a critical tool for optimization of drugtherapy. Accuracy of laboratory testing to supportTDM depends on a deliberate coordination of bothpre-analytical and analytical sources of interferences.Timing of specimen collection, performance character-istics of analytical techniques, and patient historyguide interpretation of a TDM result. Any result that isinconsistent with clinical expectations should becritically evaluated before dose adjustments are made.

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