GENERAL RECOMMENDATIONS FOR THE ESTABLISHMENT OF METHODS TO MEASURE IMMUNOSUPPRESSIVE DRUGS
The life cycle of a measurement procedure can be divided into distinct phases. If a clinical need can be defined (eg, to keep a patient within a recommended drug target range), analytical goals can be formulated (eg, assay range, requirements for analytical precision, etc.). Based on such quality requirements, a rational measurement platform and method selection leads to the development or establishment of a method. Once a stable method is available, performance goals are transformed to a list of validation benchmarks that are tested by appropriate measurements. Data derived from these measurements are compared against the predefined performance goals. If all goals are met, the implementation of the measurement procedure can be undertaken. If not, the method design has failed and must be modified. Once implemented, an assay must be monitored by appropriate QC procedures (eg, system suitability testing,94 internal QC, periodical reevaluation of test performance, proficiency testing, etc.). Whenever possible, the measurement platform life cycle should be accompanied by a risk assessment that has been initiated in the design phase of the project.
Meaningful method design must meet clinical needs. The analysis frequency, time to report results, laboratory workflow issues, desired analytical range, and minimal requirements for assay precision (ie, derived from biological variation and the impact on clinical decision-making) are the cornerstones for measurement method selection. For ISDs, this means that a total turnaround time of 3–6 hours is desirable in a transplantation center setting to allow for daily dose adjustment in acute situations. In particular, for LC-MS/MS platform-based analytical services, this can be challenging. If only outpatient units or external care providers have to be serviced or tied to the reported results, overnight reporting can be sufficient. Characteristics specific to individual ISDs that should be considered when designing an analytical method are given in Table 1. Frequently used comedications such as other ISDs, antibiotics, antifungals, antivirals, antidiabetics, antihypertensive drugs, lipid-lowering agents, and nutritional supplements can be extracted from the respective prescribing information.
For In Vitro Diagnostics (IVD)-CE certified or FDA cleared commercial tests, the producer must clearly state which guideline was followed for method validation. For a LDT, this documentation must also be given. Furthermore, it must not be forgotten that hardly any of the guidelines referred to above allow complete assay validation for clinical use. Therefore, a validation plan needs to be internally developed and should consider national regulations, the type of methodology, the targeted immunosuppressive drug, its physicochemical properties and specific clinical requirements. The written validations plan must be clearly defined as such; the validation experiments need to be described in sufficient detail and predefined acceptance criteria should be stated. For an overview of key validation elements deemed necessary to characterize a bioanalytical method, the reader is referred to the literature95–98 and to the aforementioned guidelines. Specific additional recommendations for ISD TDM methods are summarized in the following sections.
Method validation can be either a full validation or a partial validation. A full assay validation should always be performed prior to the clinical application of any newly developed method and of any method setup based on literature data. The primary performance characteristics essential to demonstrate the reliability of a method include the measurement range (including the lower [LLOQ] and upper limit of quantification [ULOQ]), assay precision and accuracy, specificity for the parent drug, robustness against interference and pitfalls specific to the chosen technique, carryover, and stability of the analyte, the sample extracts and reagents under storage and processing conditions. If the storage conditions for assay calibrators and controls (frozen) differ from those of the patient samples (fresh), investigation of fresh and thawed aliquots of identical specimens to control for calibration bias due to matrix discrepancies between thawed calibrators or controls and fresh patient samples should be part of the validation protocol as well. Moreover, if applicable, issues such as the suitability of lyophilized QC materials and calibrators used in an analytical procedure, the effects of collection tube separating gels, risk of nonspecific adsorption to surface materials (eg, glass, PE, PVC, etc.) or the use of different suppliers or different lots of reagents and other materials must be addressed during validation. When minor changes have been made to an assay (as transfer to another instrument of the same model, a change in equipment and reagent provider or a change in storage conditions, etc) a partial validation is necessary. This may also be required if a method has been out of control as for example indicated by failing long-term quality tracking acceptance criteria or proficiency testing, and if a previously validated assay has not been used for an extended period. The scope and extent of a partial validation depends on the modifications and issues requiring revalidation and may vary from evaluating a single performance characteristic to an almost full validation.23,99
Even when IVD-CE–certified or FDA-cleared commercial procedures are used, laboratories must verify data on the assay performance and assay specific target ranges given by the manufacturers, and their conformity to both analytical and clinical requirements. In addition, conformity to certification or clearance requirements (eg, the completeness of the package insert—does the intended use match with the population to be monitored?) should be reviewed. A summary of the performance characteristics of presently available assays as reported by the manufacturers is shown in Table 2. As mentioned above assay parameters reported by the manufacturer might be different from those observed by the users during routine application.
Validation/verification of methods for ISDs should be performed on samples based on the same matrix and the same anticoagulant that are intended to be used in routine services. Whole-blood samples are recommended for the TDM of cyclosporine, tacrolimus, sirolimus, and everolimus, whereas plasma is the material of choice for mycophenolic acid (Table 1). Ethylene-diaminetetraacetic acid (EDTA) is the preferred anticoagulant because it minimizes problems with clotting and its use allows for the quantification of multiple immunosuppressive drugs in the sample. Method validation/verification should include experiments with actual patient samples because they reflect the relevant proportions of free and bound drug and of parent drug and drug metabolites. In addition, testing for the effects of comedication and disease conditions should be performed. Spiked samples could be used to supplement the experiments, but one should be aware that they may not provide an accurate assessment of the performance characteristics and demonstrate the robustness of the method if used as the only matrix. Furthermore, it is recommended to include fresh patient materials to the sample set because frozen samples do not allow for the full testing of the sample pretreatment procedure. For instance, sample pretreatment for analysis of cyclosporine, tacrolimus, sirolimus, and everolimus should ensure complete hemolysis of fresh whole-blood samples, but this has already occurred when the whole-blood samples have been frozen. As usual, it is important to make sure that instrument/system suitability testing is conducted regularly during method validation and verification.
It has to be noted that assay validation can potentially become a self-confirming system and that even after meeting all acceptance criteria, results might be incorrect. This can particularly occur if in-house calibrators have been used that have incorrectly been serially diluted. Hence, measurement of external controls and crossvalidation with peer laboratories is critical before the measurement of clinical TDM samples can commence. Whenever laboratories interchange different validated and/or verified methods performed on qualified platforms, for example, when moving a measurement service between different models of an instrument (eg for back-up purposes) an assay crossvalidation assay is required. Method crossvalidation should include the analysis of both QC and patient samples. Patient samples should mirror the monitored population.
All results generated during the method validation/verification should be summarized in a validation report which includes at least information about the experiments performed, the materials and reagents used including calibrators and QC samples, the source of the specimens used, the statistical analysis performed, the analytical performance and the acceptance criteria used for approval. Unexpected results obtained during validation/verification and deviations from the recommended performance must be discussed with regard to their significance to the clinical dose adjustment process.
METHOD PERFORMANCE CHARACTERISTICS TO BE INCLUDED IN THE VALIDATION AND ACCEPTANCE CRITERIA
As mentioned above, validation procedures for methods designed to provide ISD TDM services should follow the detailed procedures described in the guidelines of international scientific societies and governmental agencies whenever feasible. Hence, the following paragraphs will focus on specific issues related to ISD analysis, rather than recapitulating these well-known guidelines. Earlier, IATDMCT recommendations regarding acceptance criteria for analytical method performance have been updated to reflect current clinical needs and technical advances.8–12,14,39
Method Specificity for the Parent Drug
The target ranges currently specified by TDM of ISDs are for the parent drug. This is either because of a lack of significant pharmacological activity of drug metabolites or because of a negligible concentration of pharmacologically active metabolites in the specimen compared to the parent drug5,7–11,13,14 (Table 1). Therefore, analytical methods should be specific for the parent drug determination. If metabolites are present, assay crossreactivity to these analytes should be reported to the user with a statement of clinical relevance. Crossreactivity with drug metabolites determined by a ligand-binding assay leads to overestimation of the drug concentration and is likely to cause too low drug exposure if not taken into account at dose adjustment. No current evidence exists to support the monitoring of single metabolite concentrations of any ISD as a part of a routine TDM service.
In general, even if metabolites do have an ascribed pharmacological activity, analytical methods must be substance specific. If the presence of a particular metabolite should be covered in a specific situation (eg, the acyl glucuronide of mycophenolic acid) its concentration must be evaluated as a separate analyte. That implies that in chromatographic assays, even if mass spectrometry is used as a detector, baseline separation of the chromatographic peaks and individual calibration curves for both analytes are necessary to avoid in-source fragmentation-related bias, particularly if time pressure leads to rapid chromatographic approaches.100 For ligand-binding assays, the situation is more complicated. Even if 100% crossreactivity of the capture protein to the parent drug is claimed and its bioactive metabolite has been tested in a quantitative manner by the manufacturer during assay validation (which is not the case for any currently available immunoassay for an immunosuppressive drug, Table 2), such results are likely to not be transferable to routine samples, as recently shown for the endogenous analyte 25-OH-vitamin D.101,102 Hence, results from crossreacting ligand-binding assay must not be seen as the sum of the individual levels of drug and metabolite. On the one hand, even if a metabolite is reported to be pharmacologically active, the in vivo PD and toxicodynamic properties are not necessarily identical with those of the parent drug. Conversely, crossreactivity in an immunoassay is mostly concentration dependent, is not parallel to the parent drug–response curve, and will be affected by factors, such as the metabolization rates, drug–drug interactions, time after dosing and/or clinical disease that affects drug elimination.103 However, knowledge of the expected relative concentrations of a crossreacting metabolite to the concentration of the drug at the sampling times used in the TDM (Table 1) is important during method development to estimate the potential impact on patient results. To evaluate crossreactivity with the parent drug, either spiked samples with highly purified metabolites at the highest clinically relevant concentrations can be used or the metabolites present in the patient specimen can be measured chromatographically, followed by an estimation of their effects on the immunoassay under evaluation.
When developing an analytical method, a working range (the range between LLOQ and ULOQ) that covers the full concentration range expected for each single ISD in patient samples should be aimed for. This concentration range depends on the therapeutic scheme (eg, applied dose or cotherapy) and the TDM strategy (predose concentrations, Cmax, C2, abbreviated or full AUCs). Laboratories should characterize the working range of their method, and if dilution is required to allow high concentrations to be measured, appropriate protocols should be developed and validated. Patient samples with high drug concentrations or spiked whole blood can be diluted. Because the crossreactivity of drug metabolites in immunoassays may be concentration dependent, including actual patient samples for the evaluation of dilution integrity is strongly encouraged if ligand-binding assays are to be used. To be acceptable, dilution integrity should demonstrate accuracy and precision within set criteria (see below). A standard procedure for the reporting of concentrations outside the working range should be available. It must not be overlooked, that in general some immunoassay designs may lead to a “high-dose hook effect” in which the apparent analyte concentration drops to almost zero at very high concentrations.104 A manufacturer must report to the user about affected products in the product information.
The LLOQ issue is particularly important because very low target ranges for some immunosuppressive drugs have been introduced in an attempt to reduce long-term toxicity.105 Hence the quantification limit must be geared toward the lower limit of the target range that can be understood as a decision limit and not vice versa. To ensure a reliable determination of such low concentrations and to detect inappropriate low dosing or patient nonadherence, analyte quantification should be at least one-third to half of the lower limit of the target concentration window, for example, an LLOQ close to 1 mcg/L should be achieved for tacrolimus, sirolimus and everolimus assays to allow meaningful TDM at 2–3 mcg/L drug concentrations. Similarly, LLOQs of 20 mcg/L for cyclosporine and 0.2 mg/L for mycophenolic acid procedures should be targeted. Method imprecision and an inaccuracy of ≤20% at the LLOQ must be demonstrated during validation or revalidation, as stated in several guidelines.
Measurement of free or intracellular drug concentrations is currently not established or recommended for TDM services for any immunosuppressive drug. However, some reports demonstrated promising results with such strategies,106–108 and the high sensitivity of modern LC-MS/MS instruments enables such analyses. Clearly, a lower LLOQ than those given above would be necessary to quantify free or intracellular ISD concentrations.
Reproducibility of the results is important to facilitate consistent dosing decisions. Both within-run and between-run precision should be characterized using concentrations corresponding to the within, above, and below the recommended target range defined for the respective single drug. In general, for ISD methods, a CV of ≤10% or even ≤6% (see total analytical error estimate calculation further below in this section) should be aimed for. This requirement refers to between-day imprecision and is based on estimated variations that may result in poor therapeutic decisions.109,110 It has to be noted that these values are more restrictive than CV ≤15% presented in the EMEA and FDA guidelines,22,23 which are however not targeting clinical routine measurements.
Measurement accuracy, the closeness of agreement between a single result and the true concentrations of the analyte,21 has recently received particular attention because a large number of different assays have entered the marketplace. Although they are reproducible, many of these assays generate distinctly different results due to crossreactivity or improper calibration, which is confusing in a clinical setting. Hence, availability of methods that produce unbiased concentration measurements is an important issue. From a clinical viewpoint, efforts should be made that patient samples can be tested with any validated/certified method in any laboratory without any impact on the dosing advice.
Accuracy can be evaluated by means of an analysis of materials with an assigned drug concentration (eg, third-party prepared calibrators, or controls traceable to a well-characterized reference material) and through a comparison with a validated reference method. In addition, the results generated with the method during validation can be compared with those from external proficiency testing. As mentioned above, using authentic patient samples to assess accuracy is essential to uncover potential specimen-related bias (eg, due to matrix effects or crossreactivity to metabolites). Although external “pooled patient” proficiency-testing samples are very useful, they can also underestimate errors that may occur in individual patients because pooling blood tends, on average, to decrease the magnitude of error that might occur for individual patients due to matrix effects. In addition, proficiency-testing samples are typically prepared using leftover material from previous TDM analysis that has sometimes undergone multiple freeze–thaw cycles and/or has been stored under poorly controlled conditions, compromising sample integrity. Therefore, evaluation of the performance of new assay measurements using a chromatographic reference method and actual (nonpooled) samples is advised when first working with patient samples, and when large individual deviations are suspected.39
Before an exact-matching isotope-dilution mass spectrometry method reference service is available for ISDs, fully validated LC-MS/MS–based procedures with well-documented assay performance that are specific for the parent drug should be considered as the reference.111,112 However, both data from external proficiency testing and data from the literature demonstrate distinct differences in the performance of individual LC-MS/MS assays.111 In addition to a lack of standard reference methods, comparison of analytical procedures is hampered by the absence of certified reference materials. Tacrolimus is the only ISD for which a whole-blood certified reference material is available (ERM-DA110a), and two studies have already shown that this reference material can be used to test the accuracy of methods, and that standardizing the individual laboratory-developed LC-MS/MS procedures can help in minimizing between-method bias originating from a lack of interassay accuracy.111,112
Methods should be compared by an unbiased procedure as Deming regression or Passing Bablok regression.113,114 A comparison should include samples from a wide variety of pathologic conditions that are characteristic for the intended patient population (different transplant types, time post-transplantation, time of blood draw with respect to drug administration, ethnic backgrounds, age groups, etc.) and present a wide range of concentrations including those within, above, and below the recommended target range. In some cases, a separate analysis of specific subsets of the data may be indicated. For example, when verifying the performance of an immunoassay, a separate analysis of data for each organ transplant group gives a better estimate of the effect of drug-to-metabolite ratios that may be due to differences in the formation or elimination of crossreacting metabolites.
Meeting the following criteria compared to a reference procedure is recommended for the acceptability of a method for the selective determination of ISDs:8,12,14
- A linear regression slope within ±10% of the theoretical value of 1.0.
- A linear regression intercept not significantly different from zero.
- A standard error for the estimate, Syx ≤10% of the average of the target concentrations.
The number of samples tested must be sufficient to allow for meaningful statistical analysis (n = 40–100), and comparison measurements must comprise several calibration events (n ≥ 3), allowing for an assessment of the intermediate accuracy in the data evaluation. In addition, a data evaluation for the comparison of measurements must include an absolute and relative Bland–Altman plot and an evaluation of the individual measurement bias to ensure that outliers are treated appropriately.115 Such treatment might include repeated measurement on both platforms to exclude measurement error; investigation of other clinical parameters, including hemolysis, icterus, and lipemia; plasma exchange experiments to screen for interfering molecular entities; and medical record investigations to evaluate comedications that might, for example, interfere with the metabolism of the monitored drugs and clinical condition (eg, impaired renal function) of the patient.
Precision and trueness (bias) of ISD-TDM methods must be assessed by comparison with meaningful clinical and physiological target ranges.116 The drug concentration varies with the state of the patient, so target ranges immediately after transplantation may differ from those for long-term transplant patients. Because data on longitudinal intraindividual biological variation of ISDs are still scarce,117,118 it should be feasible to estimate the analytical variability and bias from clinical TDM goals, for example, to determine a trough concentration range for a patient. If, for example, a tacrolimus target range is set from 8–10 mcg/L, it can be assumed that this range mirrors the intraindividual biological variability for a patient to be clinically meaningful. Because individual measurement points in this range are usually following a Gaussian distribution, this range can be interpreted as a 95% CI (“2S range”) around a mean drug concentration of 9 mcg/L. Consequently, the CV associated with this intraindividual biological variability can be assumed to be CVI = 5.6%. Applying the widely accepted model by Fraser et al (CVA < 0.5 × CVI)119 that derives the desirable analytical imprecision from the intraindividual biological variability,120 a CVA = 2.8% is calculated. Such a CVA is barely achievable in routine use. Even with a broader target range, for example, 6–12 mcg/L for tacrolimus (CVI = 11.2%) or 85–135 mcg/L (CVI = 11.2%) for cyclosporine, the derived CVA = 5.6% is a challenging analytical goal. Applying the more TDM-related CVA model by Glick121 (CVA < 0.1*(CU−CL)/CU, where U and L are the upper and lower range limits) that focuses on the therapeutic target range as such and is based on the assumption that the analytical error must be significantly smaller than this range, the CVA to be aimed for is of a similar size (CVA = 2.0% for the range 8–10 mcg/L; CVA = 5.0% for the range 6–12 mcg/L). If the analytical performance goal estimation for TDM by Fraser18,122 is applied, which is based on fundamental PK theory, the ISD-TDM CVA values must also be much below 10%, depending, of course, on the half-life and the dosing regimen for the individual drug. In general, the discussed error estimate for tacrolimus agrees with data for other TDM analytes with similar target ranges.123,124 Translating the analytical imprecision goal CVA = 5.6% into a total analytical error estimate (TEA = 1.65* CVA + BA), the allowable analytical bias BA must be lower than 5.8% if a TEA goal of 15% is assumed to be feasible. Again, this is a challenging goal particularly if interassay bias values for the ligand-binding assay from metabolite crossreactivity or for laboratory-developed mass-spectrometry installations from calibration heterogeneity are considered (Figs. 1 and 2). In summary, for ISD TDM, a CVA of approximately 6% should be aimed for. Whenever BA exceeds the lower percentage range (to be expected if calibration systems depending on different reference standards are used), TEA will increase such that it is extremely difficult to monitor ISDs even if a clinically rather large target range (eg, 6–12 mcg/L for tacrolimus) would be accepted.
Specific Technique-Related Pitfalls and Interference
Method validation should include an evaluation of specific technique-related pitfalls, which might cause errors in drug concentration estimates. To test for such effects, sample drug concentrations should be adjusted to near the medical decision limit. Examples for immunoassays are interferences due to crossreactivity with other drugs and metabolites, reactions with heterophilic antibodies, antibodies directed against the binding antibody, and the effects of endogenous factors, such as hematocrit, albumin, bilirubin, and triglycerides.125,126 For instance, the results generated by the tacrolimus IMX II-MEIA, which has now been removed from the market, were shown to be strongly affected by the sample hematocrit.117,127,128 A number of false-positive results have been reported for immunoassays based on the ACMIA test format, most probably due to the problems with heterophilic antibodies.129–131 For LC-MS/MS procedures, a major pitfall is the possible presence of matrix effect (eg, ion suppression or ion induction) that is particularly risky when using electrospray ionization (ESI).132–134 Both the effects of hydrophilic and lipophilic matrix components as salts or phospholipids, respectively, are an issue.135,136 High extraction and chromatographic separation efficacy (eg, using SPE or 2D-chromatography) and the use of stable isotope internal standards can minimize matrix effects.135 Other possible problems that can compromise results and that must be addressed during the validation of LC-MS/MS procedures include in-source fragmentation (demonstrated, eg, by the glucuronide metabolite of mycophenolic acid glucuronide [MPAG]81), interference with internal standard transitions by drug metabolites (eg, when using cyclosporine D as an internal standard for cyclosporine analysis61) or interference with drug analysis because of contamination of the internal standard (eg, the analysis of sirolimus in a multiplex assay using 13C2D4-everolimus as the internal standard137). In addition, factors such as isotopic purity issues, cross-talk between MS/MS channels, and isotopic integrity should be considered while validating a LC-MS/MS method.
Both analysts and clinicians should be aware of possible pitfalls of the methods used when interpreting patient results.135 The laboratories providing services for TDM of ISDs are responsible to continuously inform/educate their clinical partners about issues that might affect the performance of a particular method.
Carryover can occur during analysis and compromise accurate measurements. Therefore, it should be evaluated as part of the validation of measurement procedures for ISDs. If carryover is detected in a blank sample processed immediately after a sample with a drug concentration close to the highest expected clinical concentration, it should not be greater than 20% of the LLOQ.
As previously mentioned, method validation should include an assessment of the short-term and long-term sample stability and the stability of stock solutions, sample extracts, and reagents. Stability experiments should mimic the local conditions under which samples are collected, transported, stored, and processed as closely as possible. Expiration dates should be clearly marked and analysts should strictly adhere to those. Laboratories are advised to setup specific procedures for temperature control during the transport, storage, and handling of samples (Table 1).
Effects of Changing Solvent and Reagent Suppliers or Lots, Tubes, or Vials
Changing solvent and reagent suppliers or lots, tubes, or vials may compromise analytical reliability; therefore their quality should be always demonstrated before use for patient sample analysis.
Dried Blood Spot Sampling Specific Issues
DBS represents a new intensively investigated strategy for the TDM of ISDs to facilitate outpatient management.138 However, this sampling strategy has some specific challenges139,140 that should be considered during method validation. The major drawback is that the volume of the blood sample cannot exactly be measured. Thus, calculation of ISD concentration is based on the assumption that the punched out filter paper blood spot is saturated and always contains the same blood volume. This assumption may not always be correct. Important factors that may affect the accuracy of the results include sampling practice, the size and drying time of the spot, the temperature and humidity of the environment, the type of Dried Blood Spot (DBS) paper and the on-card storage stability. Hematocrit141 plays a particularly significant role for DBS because different hematocrit values are associated with different viscosities, impacting on the spread of the blood on the DBS paper. As shown by Koster et al,142 the specific combination of DBS samples with extremely high drug concentrations and extremely low hematocrit values is challenging. For specifics related to validation of methods for DBS, the reader can refer to the recommendations of the European Bioanalysis Forum.143
METHOD LIFE-CYCLE MANAGEMENT
The long-term consistency of the results generated with a method is of high importance in transplantation medicine. ISDs are used in life-long treatment of most transplant patients; individualized target ranges are usually established post-transplantation with a certain method (eg, LC-MS/MS at the transplantation center). Usually patients stay attached with their transplantation center for several years with visiting time intervals from some weeks to several months. Hence, the ISD-TDM platform must be stable over such times; any long-term inconsistency of results may negatively impact dosing decisions and the patient outcome. Therefore, a method life-cycle management should be established to guarantee that analytical performance documented during method validation is continuously reproduced.
Stable analytical performance over time is based on a robust assay performed by well-trained personnel on well-maintained instruments. Assay bias and assay imprecision are kept low by monitoring the assay while it is being performed and by following good laboratory practices. Measures to avoid calibration bias include participation in an external quality assurance program, the use of external commercial calibrators, QC materials (preferably from different manufacturers), and the availability of certified reference materials and reference methods for all ISDs.
A rigorous internal quality assurance program that includes both system suitability testing (control of temperature in different compartments of the instrument, signal accuracy, signal stability signal intensity, signal recording, retention times, etc.), and revalidation of critical analytical parameters for existing methods is strongly recommended. Laboratories should have established protocols for these procedures that should be conducted on a regular basis to ensure continuous fitness with regard to analytical specifications and clinical requirements. For instance, the introduction of new therapies to the intended patient population or extension of this population to new disease groups may introduce challenges that were not present during the initial method validation. For example, experience from the Symphony clinical trial144 demonstrated that although the intended tacrolimus concentrations were in the range 3–7 mcg/L, most patients had actually been at or above the top concentration in this range. This was partly because the analytical method used by most centers to measure the drug (MEIA) was never designed to measure tacrolimus at such low concentrations.145
Guidance on how to design and implement an appropriate quality assurance program is usually provided by national laboratory regulations, such as the Clinical Laboratory Improvement Amendments (CLIA) in the US or the RiliBÄK (Quality Standards for Medical Laboratories of the German Chamber of Physicians) in Germany.146 Furthermore, ISO15189 in its current version (ISO15189:2012, version 2014-08-15) specifies requirements for the quality and competence in a medical laboratory. The QC strategy of every laboratory should be to determine when a method is out of control, the reasons why, and when it returns under control. For example, a plan including types and levels of QC materials to be run, frequency of performing QC runs, QC acceptance criteria, and statistical evaluation of QC results should be established to reliably detect both systematic (trends or shifts) and random errors. Appropriate control levels should concentrations within, above, and below the recommended target ranges with the lowest concentration not exceeding 3 times the LLOQ. Audits and reviews aiming for improvement should be conducted on a regular basis to allow for timely detection of deviations from the specifications. All procedures and measures to monitor, evaluate, and guarantee stable performance of the analytical measuring system, as well as interventions to resolve “out-of-control” situations should be carefully documented. Profound documentation serves a dual purpose: it documents the performance of the assay (particularly long-term) and will facilitate troubleshooting.147,148
In addition to the internal quality assurance program, laboratories performing ISD TDM must participate in an external quality assurance program to allow continuous crossvalidation and proof of analytical quality (eg, Analytical Services International Ltd,91 CAP149). In addition to spiked samples, proficiency-testing samples should include samples that do not contain the drugs of interest and particularly pooled transplant patient samples from patients after the transplantation of different organs, as well as from nontransplanted patients, if applicable.
Continuous education and training of TDM laboratory personnel is an integral part of ensuring a high level of analytical quality. Therefore, establishing programs to maintain an adequate educational and training level of the personnel involved in analysis and reporting or interpreting the results is strongly recommended. Finally, it should be reminded that the clinical effectiveness of TDM (of ISDs particularly) largely depends also on the respect of the sampling hours and of any preanalytical recommendations as correct sampling from catheter sytsems.150,151 Continuous education should therefore ideally include the nursing staff and health care professionals.
Reliable performance of analytical methods that are used for TDM services focusing on immunosuppressive therapy in transplantation is essential to enable proper therapeutic decisions and dose adjustment. This document was developed on behalf of the Immunosuppressive Drugs Scientific Committee of IATDMCT. It aims to provide recommendations for the establishment and maintenance of appropriate laboratory practices, to adequately reflect current clinical needs for advanced optimization and individualization of the therapy with ISDs to allow for prolonged graft survival and an improved quality of life of organ recipients. These recommendations address all phases of the analytical procedure life cycle, including method design, method validation and performance verification; the definition of appropriate acceptance criteria for analytical performance; risk assessment; and method life cycle management. Regarding method validation, a proposal on how to adapt the recognized guidelines published by international scientific societies and governmental agencies, for the analysis of ISDs, has been provided. Both specifics related to LDT and commercial tests at the analytical site are covered. Actions aimed at improving the consistency of the reported results (including between-method, between-laboratory, and over time consistency) have the highest priority. These include but are not limited to the advanced standardization of methods and testing practices, the development of appropriate reference materials and reference methods, the improved availability of actual patient sample-based materials from external proficiency testing, the enhanced level of compliance to internationally accepted laboratory practice guidelines and to the defined TDM-specific recommendations through rigorous education and continual training. We are convinced that at the current state especially LC-MS/MS–based LDT systems clearly need methodological consolidation and guidance. Possible new areas of development, such as the measurement of intracellular concentrations, new sampling strategies, method multiplexing, or point of care testing, may need to be further elaborated in the current recommendations in the future to cover specific requirements if clinical implementation is intended.
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Keywords:Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
TDM; immunosuppressant drugs; cyclosporine; tacrolimus; sirolimus; everolimus; mycophenolic acid; LC-MS/MS; immunoassay; design; validation; verification