August 2011: Volume 37, Number 8
An Update on Therapeutic Drug Monitoring
By Susan Maynard, PhD, FACB
Worldwide, patients undergo 50,000 solid organ transplants each year, more than half of which are kidney transplants. Other solid organs transplanted include liver, heart, lung, pancreas, and intestine. Early efforts to prevent acute rejection of the transplanted organ focused on immunosuppression. As a result of these efforts, the 1-year survival rate, an indicator of acute rejection, dramatically improved. The greatest improvements came with the introduction of cyclosporine in the early 1980’s, followed by tacrolimus, another calcineurin inhibitor, and later by sirolimus and everolimus, both mammalian targets of rapamycin (mTOR) inhibitors.
While the initial hurdle in solid organ transplantation was to conquer acute rejection, today the challenge is improving long-term organ and patient survival, without compromising acute rejection rates. Chronic allograft injury affects all transplanted organs and with an incidence of 39% is the most common cause of graft loss after the first year (1). But almost as many patients with renal transplants died with a functioning graft, 31% (2). Infection, malignant conditions, and cardiovascular disease accounted for 11.7%, 10.1%, and 30.1%, respectively, of these deaths with a functioning renal graft. Overall, the death rate in renal transplant patients caused by each of these conditions exceeds the death rate in similar nontransplanted patients, implicating immunosuppression complications.
Not surprisingly, new concepts in the field of immunosuppression are very often related to lowering immunosuppressant levels in order to diminish the side effects of immunosuppressant drugs. This review will focus on the laboratory’s role in increasing the long-term survival of transplant patients through improvements in therapeutic drug monitoring (TDM) of immunosuppressants.
Balancing Toxicity and Immunosuppression
Today, clinicians are employing combined therapies of a calcineurin inhibitor and mTOR inhibitor to permit lower doses of each type of immunosuppressant, thereby decreasing the toxicity of both drug classes. Sometimes, mycofenolate mofetil, an antiproliferative drug, can be added to the regimen to allow a lower dosage of the other immunosuppressants.
TDM of immunosuppressants seeks to balance the toxicity of immunosuppressant agents against adequate immunosuppression to prevent rejection. On the toxic side of this balancing act the players are: cardiovascular disease, diabetes, hypertension, hyperlipidemia, infection, nephrotoxicity, neurotoxicity, malignancy, and metabolic bone disease. These side effects often contribute to cardiovascular disease and occur frequently. For example, calcineurin inhibitor nephrotoxicity is virtually universal at 10 years post transplant (3).
Pharmacokinetics and Pharmacogenetics
Absorption and metabolism of immunosuppressants vary both intra- and inter-individually. Both calcineurin inhibitors and mTOR inhibitors are oxidized by cytochrome CYP3Ain the liver and intestinal wall. The expression of CYP3A activity and the subsequent P glycoprotein transport from the intestinal wall to the liver are genetically determined. Researchers also have linked the expression of CYP3A activity to ethnic groups. Approximately 15–25% of Caucasians and 70% of African Americans express CYP3A5 activity. These variations in CYP3A5 activity can result in as much as a two-fold increase in tacrolimus clearance, causing an expresser of the enzyme to require a dosage of tacrolimus 2–3 times higher than a non-expresser (1).
Other factors also contribute to intra- and inter-individual variability in drug bioavailability. These factors include: changes in liver function; bile flow; diarrhea or gastrointestinal surgery; concomitant treatment with other drugs metabolized by the P450 system; changes in immunosuppressant product formulation; time post transplantation; and age, with pediatric patients metabolizing immunosuppressants more rapidly than adults. Diet also affects immunosuppressant absorption, with higher fat intake leading to less absorption, and foods like grapefruit juice that affect the P450 system resulting in altered metabolism. The timing of any food intake in relation to the timing of medication also can affect immunosuppressant concentrations. For all these reasons, drug bioavailability can vary by 5–40%.
Given the tight window that separates adequate immunosuppression from toxicity, it is imperative that patients have their drug levels monitored and dosages adjusted.
Of course, analytical issues for therapeutic immunosuppressant monitoring remain a critical concern for the lab. Sirolimus stored at >30ºC lacks stability, making sample transport difficult. Early in the development of analytical methods, a major issue with cyclosporine, and later tacrolimus and sirolimus analysis, was the analysis matrix: serum or plasma versus whole blood. Matrix is an issue with these drugs because temperature fluctuations can cause them to bind to red blood cells differently. In 1990, multiple consensus forums agreed whole blood was the sample of choice.
Another early issue with immunosuppressant analysis was whether to assay parent drug or parent plus metabolites. In the bloodstream, each of the immunosuppressant drugs is transformed into multiple metabolites. Some of the metabolites possess immunosuppressant properties, while others are nephrotoxic. Furthermore, the proportion of metabolites to parent may change, especially with changing liver function. Based on recommendations from the consensus forums, laboratories established the monoclonal parent compound as the appropriate target for analysis. The recommendations also made target levels more organ-specific and similar from institution to institution, although some inter-institutional variation based on methodological differences still remain today.
Unlike older heterogeneous competitive binding methods, newer antibody-based methods of measuring immunosuppressant drugs do not require separation of bound from unbound drug. Many assays also are now available on clinical chemistry analyzers that are routinely part of core laboratory instrumentation. These homogeneous methods include: affinity column-mediated immunoassay (ACMIA); chemiluminescent microparticle immunoassay; cloned enzyme-donor immunoassay; direct chemiluminescent immunoassay; enzyme-multiplied immunoassay; fluorescence polarization; and microparticle enzyme immunoassay (MEIA).
One issue shared by all of the competitive binding methods is cross reactivity with either metabolites or heterophilic antibodies. In general, cross reactivity causes immunoassays to have a positive bias compared to chromatographic methods. When using an antibody-based assay, it is important to know the cross reactivity, which can be found in the manufacturer’s package insert. Tables 1–3 provides the cross reactivities of different immunoassay methods for cyclosporine, sirolimus, and tacrolimus along with their bioactivity and concentration relative to parent compound. Laboratories should be aware that not all cross reactivities have been identified and that concentrations relative to parent compound may vary, particularly with liver impairment.
Click here for Tables 1–3
Another helpful tool to examine cross reactivity is proficiency testing results on pooled samples. Figure 1 shows box-and-whiskers plots for cyclosporine, sirolimus, and tacrolimus on pooled bone marrow or liver transplant samples.
Proficiency Testing Results
This figure shows the inter-institutional precision of each method and the number of participants by method.
Abbreviations: ADVIA, ADVIA Centaur; CMIA, chemiluminescent microparticle immunoassay; HPLC/MS, high performance liquid chromatography/mass spectrometry; ACMIA, affinity column-mediated immunoassay; CEDIA, cloned- enzyme donor immuunoassay; EMIT, enzyme-multiplied immunoassay; FPIA, fluorescence polarization immunoassay; RIA, radioimmunoassay; and HPLC/UV, high performance liquid chromatography/ ultra violet detection.
Source: Analytical Services International, Ltd. Used with permission. (Available online.)
There are two other immunoassay issues, both relating to extraction, that are specific to the particular antibody methodology used. For example, MEIA methods are known to be affected by hematocrit. Laboratories have observed as high as a 20% false elevation in sirolimus levels when hematocrit decreases by 20%. Furthermore, the percentage difference caused by a decreased hematocrit is greater in the lower therapeutic range. Hematocrit interference is likely due to an inverse relationship between extraction efficiency and hematocrit (4). As expected, laboratories also see this problem with tacrolimus assays. In one study, researchers found low-positive tacrolimus results using an MEIA method in anemic patients who were not receiving immunosuppressants (5). In patients whose hematocrit values vary considerably from day to day, this issue should certainly be considered a limitation. Clinicians should be aware that MEIA levels may be useful to detect a change in immunosuppressant levels in anemic patients where a low hematocrit is a constant, but they should also remain aware that there is a positive bias with MEIA methods and samples with a low hematocrit.
Researchers also have observed profound false elevations of tacrolimus levels in 1% of samples analyzed by ACMIA, in some cases as much as 20 times above reference range values (6). They postulated that such spurious elevations were due to the presence of endogenous antibody interferences not removed by the affinity column pretreatment. The elevated levels also appear to vary over time, from individual to individual, and shift overtime with the same assay.
Automated immunoassays for immunosuppressant drugs provide greater ease of use and flexibility. The Siemens Healthcare Diagnostics Dimension and Abbott Architect automated chemistry analyzers have relatively new immunosuppressant assays for all three of the major immunosuppressants. The Dimension uses an ACMIA method with no sample pretreatment prior to running the sample and offers positive sample identification throughout the process. The manufacturer’s stated functional sensitivity for tacrolimus on the Dimension is 2.4 ng/mL. The Abbott Architect runs a MEIA method. The assay requires a slightly different pre-analytical treatment step for each of the immunosuppressants and does not provide positive patient identification. The stated functional sensitivity of tacrolimus on the Architect is <2 ng/mL. The 2009 European Consensus Conference by the Committee on Tacrolimus Optimization recommends tacrolimus target trough levels of 2–4 ng/mL, which would require a limit of quantification below the therapeutic range, ideally around 1 ng/mL (7).
When choosing an automated immunoassay method, sensitivity is obviously important, but sensitivity along with precision determines the confidence level. Researchers have reported on the adequacy of low-end precision and sensitivity for both the ACMIA and MEIA methods in the literature. Based on these findings, it would be prudent for laboratories to independently evaluate the assays in determining the acceptability of sensitivity and precision of any method before adding it to their menu.
Early chromatographic methods for immunosuppressant drugs routinely employed high performance liquid chromatography (HPLC) with an ultra-violet detector, but the introduction of electrospray and triple quadropole tandem mass spectrometers (MS/MS) began a new wave of analytical innovations. As laboratories adopted this technology, the lack of standardized, commercially available calibrators emerged as a problem. In-house preparation of calibrators for HPLC MS/MS methods presented difficulties due to the binding variability of immunosuppressants to red blood cells.
Selection of internal standards was another analytical issue in development of HPLC MS/MS methods for immunosuppressant monitoring. Commercially prepared deuterium-labeled internal standards are now more readily available. Adding individualized internal standards for each immunosuppressant to a sample analysis, however, decreases the number of repeat counts that are performed at each mass/charge (m/e) peak, because so many different m/e peaks are being scanned in the same time frame. Since fewer repeat counts can be performed at a given m/e peak, the effect of any variant count will be more pronounced. Therefore, labs that choose to use individualized internal standards for each immunosuppressant may need to inject each analyte separately.
There are other analytical considerations for chromatographic methods, some of which are particular to HPLC MS/MS, and others that are not. The possibility of ion suppression should always be evaluated in any HPLC MS/MS procedure. Laboratories should also re-evaluate a method when any step of the procedure is modified. Seemingly insignificant changes in procedure sometimes produce unexpected results, and procedures should be established to routinely review results for within run and run-to-run reproducibility, adequacy and consistency of internal standard peaks, and sensitivity of the assay. Laboratories should carefully develop procedures to insure patient identity throughout the sample extraction process, especially as numbers of samples increase. Depending upon the extraction pretreatment used, it is possible that the same relationship between extraction efficiency and hematocrit seen with MEIA may exist with HPLC MS/MS. Laboratories also should validate the separation of metabolites from parent compounds for any chromatographic method as some metabolites tend to co-migrate with a parent compound.
Non-analytical considerations related to chromatographic analysis of the immunosuppressants include cost, throughput and turnaround time, training, and provisions for downtimes. The initial instrument cost for HPLC MS/MS is quite high, but the day-to-day operating expenses are low, making total costs comparable between HPLC MS/MS and immunoassay, especially in high-volume situations where the cost of the instrument can be spread over a larger number of tests. In some labs, analysis time can become a rate-limiting step. Generally, the throughput for immunosuppressants on an HPLC MS/MS is 2–3 minutes/sample injection, a time that is comparable to immunoassay.
Today, operating an HPLC MS/MS is much more user friendly than in the past, although laboratories still should have a dedicated primary operator to run the instrument. Vendors frequently offer training programs, which are often customized to the laboratory’s needs. Manufacturers have also become more responsive to the needs of laboratories and service in a clinically relevant timeframe. The critical nature of these tests, however, requires a backup plan for planned and unplanned downtimes. Sometimes institutions that are in close geographical proximity using the same instrumentation and methodology will correlate their assays and serve as backup for each other, eliminating the added cost of instrument duplication.
In general, HPLC MS/MS methods possess lower sensitivity for immunosuppressants than antibody methods; however, the low-end precision of HPLC MS/MS has been more difficult to evaluate. Recent College of American Pathologists proficiency testing results continue to show a fairly high standard deviation for HPLC MS/MS analysis of immunosuppressants even though differences in standardization from institution to institution have been essentially eliminated. Overall, while much is known about the analytical parameters of sensitivity, specificity, and precision of both competitive binding and chromatographic analysis of immunosuppressants, these data have not been linked to patient outcomes.
The Future of Immunosuppression Monitoring
In the future, with the advent of the ability to assay dried blood spots sent directly from the patient to the laboratory, improved integration of TDM into the lifestyle of transplant patients may be possible. Dried blood spots would allow patients not only to collect samples at home without going to their physicians, but it would also mean that multiple samples timed more closely to drug administration could be collected. Moreover, analysis of dried blood spots could allow laboratories to calculate drug dosages based on area-under-the-curve or a timed post- peak level, rather than the current practice of trough levels. Both of these options are thought to represent a better way to monitor immunosuppression and predict toxicity.
Currently, neither an area-under-the-curve nor a timed-peak protocol is logistically possible, since blood draws require a trained phlebotomist. Due to the longer turnaround time, this scenario would, however, eliminate same-day dosage adjustment currently available in many physician offices. Genetic testing to supplement immunosuppressant therapy is still in its infancy, but has the potential to optimize drug selection and dosing. As mentioned previously, patients who express CYP3A can require 2–3 times higher dosages of certain immunosuppressant drugs than patients who do not. Single-nucleotide polymorphisms of this gene have been associated with nephrotoxicity.
Where is TDM Heading?
In 2011, the ideal TDM method for immunosuppressants has not yet been devised. An ideal method would offer: low sensitivity with good precision; no cross reactivity or well documented cross reactivity with limited clinical significance; low cost; turnaround time consistent with patient needs; STAT testing availability; ease-of-operation and integration into the laboratory; harmonization between institutions to allow patient mobility and consistency in research protocols; adequate back up; and infrequent downtime. No one method has demonstrated superiority in all of these aspects; therefore, laboratories should carefully select a method to meet the needs of patients and clinicians.
Until better TDM methods for immunosuppressants are available, laboratorians should be vigilant about educating users of a given method on its limitations and caution users not to use different method results interchangeably or to determine target drug levels from the literature without consideration of method differences.
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- Naesens M, Kuypers DR, Sarwal, M. Calcineurin inhibitor nephrotoxicity. Clin J Am Soc Nephrol 2009;4:481–508.
- Wilson D, Johnston F, Holt D, Moreton M, et al. Multi-center evaluation of analytical performance of microparticle enzyme immunoassay of sirolimus. Clin Biochem 2006;39:378–386.
- Taylor PJ, Morris RG. Tacrolimus measurement by microparticle enzyme immunoassay II. Ther Drug Monit 2003;25:259–260.
- Moscato D, Nonnato A, Adamo R, Vancheri M, et al. Therapeutic monitoring of tacrolimus: aberrant results by an immunoassay with automated pre treatment. Clin Chim Acta 2010;411:77–80.
- Wallemacq P, Armstrong VW, Brunet M, Haufroid V, et al. Opportunities to optimize tacrolimus therapy in solid organ transplant: report of the European consensus conference. Ther Drug Monit 2009; 31:139–152.
Susan Maynard, PhD, FACB, is technical director of chemistry, toxicology, and blood gases at the Carolinas Medical Center for Carolinas Pathology Group in Charlotte, N.C.