Accuracy evaluation of automated electrochemiluminescence immunoassay for everolimus and sirolimus compared to liquid chromatography-tandem mass spectrometry.

BACKGROUND: We evaluated the analytical performance of a newly developed electrochemiluminescence immunoassay for everolimus and sirolimus compared to that of liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: According to Clinical and Laboratory Standards Institute guidelines, the analytical performance including precision, recovery, linearity, and carryover was evaluated. For correlation evaluation, the results of Elecsys® analysis of everolimus and sirolimus were compared with those of LC-MS/MS using 120 samples from patients treated with everolimus or sirolimus. RESULTS: The within-run and total imprecision values were as follows: 2.3%-4.5% and 4.5%-6.4% for the everolimus assay; 3.3%-4.8% and 4.7%-8.1% for the sirolimus assay, respectively. The measured concentration was linear over the range of 0.718-27.585 ng/mL for everolimus analysis and 0.789-26.880 ng/mL for sirolimus analysis (all R2  > 0.99). Recovery was 93.5%-105.5% for the everolimus assay and 99.2%-109.1% for the sirolimus assay (except lowest levels). Carryover was -1.09% for the everolimus assay and -0.12% for the sirolimus assay. The results of the two chemiluminescence immunoassays showed acceptable correlations with those of LC-MS/MS (R = 0.9585 and R = 0.9799, respectively). The two immunoassays showed slightly proportional biases compared to LC-MS/MS. CONCLUSION: Elecsys® Everolimus and Sirolimus assays showed acceptable analytical performance in precision, linearity, and correlation compared to LC-MS/MS These methods can be adopted in the clinical laboratory for rapid therapeutic drug monitoring of patients who require treatment with immunosuppressants.

Clinical application of a dried blood spot assay for sirolimus and everolimus in transplant patients.

Background Monitoring of immunosuppressive drugs such as everolimus and sirolimus is important in allograft rejection prevention in transplant patients. Dried blood spots (DBS) sampling gives patients the opportunity to sample a drop of blood from a fingerprick at home, which can be sent to the laboratory by mail. Methods A total of 39 sirolimus and 44 everolimus paired fingerprick DBS and whole blood (WB) samples were obtained from 60 adult transplant patients for method comparison using Passing-Bablok regression. Bias was assessed using Bland-Altman. Two validation limits were pre-defined: limits of analytical acceptance were set at >67% of all paired samples within 20% of the mean of both samples and limits of clinical relevance were set in a multidisciplinary team at >80% of all paired samples within 15% of the mean of both samples. Results For both sirolimus and everolimus, Passing-Bablok regression showed no differences between WB and DBS with slopes of 0.86 (95% CI slope, 0.72-1.02) and 0.96 (95% CI 0.84-1.06), respectively. Only everolimus showed a significant constant bias of 4%. For both sirolimus and everolimus, limits of analytical acceptance were met (76.9% and 81.8%, respectively), but limits or clinical relevance were not met (77.3% and 61.5%, respectively). Conclusions Because pre-defined limits of clinical relevance were not met, this DBS sampling method for sirolimus and everolimus cannot replace WB sampling in our center at this time. However, if the clinical setting is compatible with less strict limits for clinical relevance, this DBS method is suitable for clinical application.

Recent advances in analytical methods for the therapeutic drug monitoring of immunosuppressive drugs.

Therapeutic drug monitoring (TDM) of immunosuppressive drugs (ISDs) with a narrow therapeutic index is an increasingly popular tool for minimizing drug toxicity while maximizing the prevention of graft loss and organ rejection. This review focuses on trends regarding analytical methods for the TDM of ISDs since 2011. The five most commonly prescribed immunosuppressive medications are critically reviewed: cyclosporine A, tacrolimus, sirolimus (rapamycin), everolimus, and mycophenolic acid. This review introduces the general background of TDM and ISDs and presents the recent developments in using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassays for the TDM of ISDs. Finally, a future perspective for these analytical methods is briefly discussed.

Therapeutic drug monitoring of immunosuppressants by liquid chromatography-mass spectrometry.

Immunosuppressant medications allow the transplantation of tens of thousands of allografts per year and consequently have great potential to decrease patient morbidity and mortality. However, some medications have great risk associated with over- and under-dosing leading to adverse effects or allograft rejection, respectively. This necessitates immunosuppressant therapeutic drug monitoring accomplished by immunoassay or liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The former's accuracy can be hindered by metabolites of immunosuppressant medications, antibodies against these medications and heterophilic antibodies. Although LC-MS/MS has superior specificity which allows it to be less susceptible to interference, this methodology lacks standardization and the necessary throughput. Recent developments in LC-MS/MS quantitation, however, include patient-friendly sample submission as dried blood spots, higher sample throughput and commercialization. Here we critically review recent LC-MS/MS publications (January 2010 to July 2015) on the quantitation of cyclosporine A, tacrolimus, sirolimus and everolimus.

Possibilities and limitations of signal summing for an immunosuppressant LC-MS/MS method.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the method of choice for quantifying small molecules in research and clinical setting. Although there is a large toolkit to increase quantification levels for LC-MS/MS, these techniques are sometimes insufficient to attain the needed limits of quantification (LOQs) or the method becomes too impractical for routine use. We examined the possibilities and limitations of signal summing, an under-utilized, easy-to-apply practice to increase LOQs for an immunosuppressant LC-MS/MS method. The limits of signal summing for everolimus were tested by running samples of everolimus at three concentrations in triplicate programming, increasing amounts of identical transitions in a constant cycle time up to the maximum number the software permitted to sum. The increase in peak area and the signal-to-noise ratio were determined. The effect on imprecision of peak areas and response ratios was evaluated by injection of a low concentration of everolimus tenfold using respectively one and five identical transitions, retaining an identical ion counting time. We compared the imprecision, LOQ, and recovery for our routine everolimus method (using one transition for everolimus and one for d3-everolimus) and an adapted method summing three identical transitions for everolimus (and one for d3-everolimus). The increase in signal was close to the theoretically expected one with a larger experimental spread for everolimus once more than five transitions were used. There was no clear beneficial effect of summing on imprecision. The adapted everolimus method showed a lower LOQ, but comparable imprecision and recovery as the routine method. Quantification levels can be improved by signal summing. No clear effect on imprecision was observed.

Long-term cross-validation of everolimus therapeutic drug monitoring assays: the Zortracker study.

BACKGROUND: This ongoing academic collaboration was initiated for providing support to set up, validate, and maintain everolimus therapeutic drug monitoring assays and to study long-term interlaboratory performance. METHODS: This study was based on EDTA whole blood samples collected from transplant patients treated with everolimus in a prospective clinical trial. Samples were handled under controlled conditions during collection, storage and were shipped on dry ice to minimize freeze-thaw cycles. For more than 1.5 years, participating laboratories received a set of 3 blinded samples on a monthly basis. Among others, these samples included individual patient samples, patient sample pools to assess long-term performance, and patient samples pools enriched with isolated everolimus metabolites. RESULTS: The results between liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and the everolimus Quantitative Microsphere System (QMS, Thermo Fisher) assay were comparable. The monthly interlaboratory variability (coefficient of variation %) for cross-validation samples ranged from 6.5% to 23.2% (average of 14.8%) for LC-MS/MS and 4.2% to 26.4% (average of 11.1%) for laboratories using the QMS assay. A blinded long-term pool sample was sent to the laboratories for 13 months. The result was 5.31 ± 0.86 ng/mL (range, 2.9-7.8 ng/mL) for the LC-MS/MS and 5.20 ± 0.54 ng/mL (range, 4.0-6.8 ng/mL) for QMS laboratories. Enrichment of patient sample pools with 5-25 ng/mL of purified everolimus metabolites (46-hydroxy everolimus and 39-O-desmethyl everolimus) did not affect the results of either LC-MS/MS or QMS assays. CONCLUSIONS: Both LC-MS/MS and QMS assays gave similar results and showed similar performance, albeit with a trend toward higher interlaboratory variability among laboratories using LC-MS/MS than the QMS assay.