Pre-analytical sources of inaccuracy in laboratory test results are discussed in detail elsewhere in this book (see Chapters 14). However, there are many pre-analytical factors important to TDM that are unique from other types of laboratory testing, and these are briefly emphasized here. In particular, the coordination of pharmacokinetic factors for the specific drug formulation(s) of interest, with the clinical status
and co-medications of the patient, will dictate the most appropriate specimen to collect, at the most appropri- ate time. In addition, specimen collection using the wrong anticoagulant and environmental factors such as heat and light can affect drug stability and alter the concentration of therapeutic drugs. TDM measure- ments are typically collected after a patient is pre- dicted to have achieved steady-state concentration from predose (trough) collections, which occur before the next scheduled dose. Steady state is achieved when the concentration of drug in the body is in equilib- rium with the rate of dose administered and the rate of elimination (after initiation of therapy, the time equivalent to at least five half-lives of the drug is needed for establishment of steady state). A random collection 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 the pre-analytical circumstances surrounding a request for TDM.
Specimen collection containers can alter TDM results by affecting drug concentration and propor- tional stability. Failure to separate cells promptly from serum or plasma may lead to in vitro metabolism, as has been observed with fosphenytoin and mycopheno- lic acid acyl glucuronide [2,3]. Serum versus plasma, variation among tube preservatives, and gel separator tubes are relevant for accurate recovery of certain
drugs or fractions of drugs. For example, heparin col- lection tubes can increase the concentration of free (not bound to proteins) drug concentration by activating lipoprotein lipase and fatty acid concentration, which will displace protein-bound drug from albumin andα1
glycoprotein [4]. Free fraction of phenobarbital, phe- nytoin, and valproic acid was shown to be elevated in serum versus plasma, whereas the free fraction of car- bamazepine and theophylline was lower in serum ver- sus plasma [5]. The proportion of free valproic acid was 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 well and increase the free fraction of drugs that are highly bound to protein (e.g., phenytoin and valproic acid).
Inappropriate dose adjustments can be made when based on free drug concentrations determined with suboptimal specimens. In addition, citrate collection tubes were associated with a decrease in the total con- centration of valproic acid [6]. More dramatically, gel separator tubes have the potential to decrease total drug recovery through adsorption of the drug to the gel material. Separator tubes were reported to decrease the recovery of cardiac drugs, tricyclic antidepressants, anticonvulsants, and antipsychotics. As such, gel sepa- rator tubes should not be used as collection tubes for TDM unless validated to be appropriate[4].
Drug stability can also be influenced by sensitivity to light or heat and may lead to erroneous TDM
• Sample preparation
• Assay design
• Assay components
• Detection prince
Pre- Analytical
Factors
Analytical Factors
Interferences
• Best specimen
• Timing of collection
• Quality of specimen
• Patient status
• Co-medication
FIGURE 13.1 Factors that affect accuracy of a TDM result.
196 13. ISSUES OF INTERFERENCES IN THERAPEUTIC DRUG MONITORING
results if specimens are stored under inappropriate conditions. Amiodarone, methotrexate, chlordiazepox- ide, carbamazepine, chlorpromazine, haloperidol, and fluoxetine are examples of light-sensitive drugs. Rapid in vitro degradation is also recognized for bupropion, busulfan, carbamazepine, lithium, and olanzapine. The consequences of in vitro degradation include falsely low results, which could potentially contribute to inap- propriate dose adjustments. Specimens containing unstable drugs should be stored at the appropriate temperature (i.e., frozen) to preserve integrity and pre- vent falsely low results. In addition, the impact of repeat freezethaw cycles on drug integrity should be carefully 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, and polydrug therapy. Food and fluid intake can alter the pharmacokinetics (absorption, distribution, metabo- lism, and elimination) of a drug by impacting gastric pH and emptying time, which can affect drug absorp- tion. Drugs that are administered orally are absorbed into the bloodstream through the gastrointestinal tract by passive diffusion if the drugs are lipid soluble or non-ionized. Factors that affect intestinal motility, hepatic blood flow, and bile flow will also have an impact on the pharmacokinetics of a drug. For exam- ple, protein intake can affect drugprotein binding for drugs and alter drug clearance. In 1987, Fagan et al.
demonstrated that high-protein diets can increase the clearance of propranolol and theophylline[7].
Fooddrug interactions will influence drug bio- availability and drug metabolism by inhibiting or inducing drug-metabolizing enzymes. For example, grapefruit juice can inhibit the cytochrome P450 iso- zyme 3A4 (CYP3A4) in the small intestine and increase bioavailability of several drugs [8]. This isozyme, part of the superfamily of CYP enzymes associated with drug metabolism, is involved in metabolism of approx- imately half of all drugs. Increased drug bioavailability can lead to enhanced drug activity and possible toxic- ity. Drug classes that are affected by this fooddrug interaction 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-lowering drugs (e.g., simvastatin). In addition, cruciferous vegetables such as cabbage and cauliflower and char- broil foods can induce metabolism of drugs metabo- lized by the CYP1A2 isozyme. Substrates of CYP1A2 include theophylline, clozapine, and olanzapine.
Alcohol consumption can also induce the CYP2E1 isozyme to increase metabolism of certain drugs, in
addition to the well-recognized synergistic effect with the pharmacodynamics of depressant drugs. Cigarette smokers tend to require high doses of many therapeu- tic drugs to obtain optimal therapy, in part due to the fact that nicotine induces isozymes CYP1A1, CYP1A2, and CYP2E1.
Genetic polymorphisms in the genes that code for drug-metabolizing enzymes can also affect an indivi- dual’s ability to biotransform drugs for activation or elimination (pharmacogenetics). For example, genetic polymorphisms can cause a patient to be a poor or slow drug metabolizer. However, the overall impact of induction or inhibition of drug-metabolizing enzymes, whether due to interacting substances or genetic pre- dispositions, depends on whether the drug substrate is activated or inactivated by the affected isozyme(s). For drugs that are inactivated by metabolism (e.g., tricyclic antidepressants and warfarin), poor metabolizers are at risk of accumulating drug and are at risk of drug- induced toxicity if standard dosing is administered.
Rapid metabolizers are at risk of therapeutic failure due to suboptimal dosing and will require higher doses to achieve optimal therapy. The opposite is true for drugs that are activated by metabolism (e.g., clopi- dogrel and codeine). Accurate TDM is an important tool for optimizing dosing under conditions of unpredictable pharmacokinetics, such as pharmacoge- netic variants and fooddrug and drugdrug interactions.
Another source of unpredictable pharmacokinetics that requires TDM to optimize dosing is patients who are critically ill. Of particular concern are patients with impaired renal, hepatic, gastrointestinal, or cardiovas- cular function. Renal disease will reduce the glomeru- lar filtration rate and reduce the clearance of drugs that are eliminated via the kidney. Renal disease will also impact drugprotein binding in patients with ure- mia, due to uremia toxins competing for drug-binding sites to albumin, and increase the concentration of non-protein-bound (free) drug and therapeutic effect.
Liver toxicity or disease will decrease production of albumin and other proteins that bind to drugs.
Hypoalbuminemia will increase the fraction of pharmacologically active drugs and may increase the risk of toxicity. Decreased liver function will reduce first-pass metabolism and also impact expression of CYP450 isozymes in general. Gastrointestinal disease or a history of bariatric surgery, malabsorptive disor- ders, or intestinal disease may impact absorption of drugs[9]. Cardiovascular disease will decrease cardiac output, tissue perfusion, drug disposition, and absorp- tion. Reduced blood flow to the liver will decrease drug metabolism.
TDM is also necessary in pregnant or nursing women to accommodate changes in maternal
physiology and to protect the fetus or infant from tox- icity. Pregnant women have increased body fat, total body water, and plasma volume, which will decrease plasma protein concentration and drugprotein bind- ing. Lipophilic drugs will distribute and accumulate in fat and decrease bioavailability. Drugs that are hydro- philic will have a lower volume of distribution in the body and may have increased clearance and decreased bioavailability in patients with excessive body fat content. Cardiac output in pregnant women is increased, resulting in enhanced drug metabolism. A general recommendation is to carefully monitor pregnant or nursing women who require drug therapy to ensure efficacy and safety for both mother and her unborn child[10].