- Email: [email protected]
Everolimus TDM using Thermo Fisher QMS immunoassay on Indiko, Beckman DxC, AU680, and AU5800 analyzers
Accepted Manuscript Everolimus TDM using Thermo Fisher QMS immunoassay on Indiko, Beckman DxC, AU680, and AU5800 analyzers
Steven HY Wong, Kamisha L Johnson-Davis, Krista Garrison, Joe D Rankin, Chro S Muhammad PII: DOI: Reference:
S0009-9120(16)30264-8 doi: 10.1016/j.clinbiochem.2016.12.003 CLB 9444
To appear in:
Clinical Biochemistry
Received date: Revised date: Accepted date:
12 September 2016 7 December 2016 9 December 2016
Please cite this article as: Steven HY Wong, Kamisha L Johnson-Davis, Krista Garrison, Joe D Rankin, Chro S Muhammad , Everolimus TDM using Thermo Fisher QMS immunoassay on Indiko, Beckman DxC, AU680, and AU5800 analyzers. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Clb(2016), doi: 10.1016/j.clinbiochem.2016.12.003
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ACCEPTED MANUSCRIPT
Everolimus TDM using Thermo Fisher QMS immunoassay on
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Indiko, Beckman DxC, AU680, and AU5800 analyzers
Steven HY Wong1,2*, Kamisha L Johnson-Davis 3,4, Krista Garrison1,
Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT
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Wake Forest School of Medicine, Winston-Salem, NC
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Wake Forest Baptist Health, Winston-Salem, NC,
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Joe D Rankin 1, and Chro S Muhammad 2,5,6
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Sulaimania School of Medicine, Sulaimania, Kurdistan,
Kurdistan Board of Medical Science (KBMS), Kurdistan
*Corresponding author – Steven H. Wong, Ph.D., Department of Pathology, Wake Forest School of Medicine, One Medical Center Boulevard, Winston-Salem, NC 27157, USA. Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Objectives: Everolimus (EVR), a mTOR inhibitor immunosuppressant approved for renal, liver and cardiac transplants. This study established the appropriate TDM performances of the Thermo Scientific QMS EVR Assay by using the Beckman DxC, followed by comparison to the Thermo
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Scientific Indiko and a previously published LC-MS/MS assay., and Beckman AU680 and
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AU5800 analyzers.
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Design and Methods: The study initially established acceptable linearity, precision and accuracy of the QMS EVR turbidimetric assay. Sample preparation was initiated by mixing patient whole
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blood with methanol and a precipitation reagent. Supernatant was transferred to sample cups.
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Drug in the supernatant and drug coated on microparticle compete for the limited number of antibody binding sites. If EVR is absent in the sample supernatant, EVR coated microparticles
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are agglutinated in the presence of antibody reagent. If EVR is present, agglutination is partially
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inhibited, depending on EVR concentration. Calibrators range was from 1.5 to 20 ng/mL. Comparison studies data were analyzed by Deming Regression and Bland-Altman plots.
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Results: Precision studies showed the following mean concentrations 4.00 – 4.72, 7.70 – 8.20
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and 14.80 – 15.56 ng/mL, and CVs of 3.1 – 8.7%, 3.4 – 8.9% and 2.6 – 4.4 % respectively. Comparison of analyses of 107 de-identified transplant samples by three analyzers- Indiko, DxC
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and AU680 showed: EVR concentrations from <1.5 to 13.6 ng/mL, slopes 1.000 to 1.076, intercepts -0.053 to 0.462, and R 0.945 to 0.981. Another series of comparison studies (n=104) of Indiko and AU680 with LC-MS/MS showed the following: slopes 1.035 to 1.086, intercepts -.019 to -0.265, and R 0.924 to 0.980. 2013-2016 CAP survey results were acceptable.
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ACCEPTED MANUSCRIPT Conclusions: Based on the experience of the past 3.5 years, Thermo Scientific QMS EVR Immunoassay using four different analyzers offered adequate limit of detection and acceptable
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accuracy and precision, suitable for monitoring renal and liver transplant recipients.
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Keywords
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Everolimus TDM, autoanalyzers, immunoassay, LC-MS/MS, performance evaluation, multi-center study, survey results
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ACCEPTED MANUSCRIPT Introduction: Everolimus (EVR) is a mTOR inhibitor immunosuppressant with FDA approvals for renal and liver transplants.1-4 EVR is often used in combination with calcineurin inhibitors (CNI) such as cyclosporine and tacrolimus (FK-506). It has also been used for cardiac transplant. The marketing names for EVR are: Zortress in U.S., Certican for Europe and other countries, and
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Afinitor for cancer treatment. EVR can also be used for treating cancers, advanced breast cancer,
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and the orphan disease tuberous sclerosis complex.5
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subependymal giant cell astrocytoma, neuroendocrine tumors of pancreatic origin, renal cancers
EVR is rapidly absorbed, with blood concentration peaking between 1.3-1.8 hr, half- life ranging
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18-35 hours, and bioavailability ranging from 5-26%. With twice daily dosing, steady state might
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be attained within 7 days.1,4 Age, weight or sex do not affect pharmacokinetics in adults. EVR is metabolized by CYP 3A4, 3A5 and 2C8. EVR pharmacogenetics was also recently and
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extensively reviewed by Johnson-Davis,6 and Picard.7 Further metabolism study employed
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quantum chemical calculations and molecular simulations which predicted region-specific hydroxylations and O-dealkylations.2 Using a combination of high-resolution TOF MS,
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hydrogen/deuterium exchange and LC-ion trap mass spectrometry, key fragmentation pathways
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of 24-hydroxy EVR were characterized, using 128 patient samples, leading to identification of other metabolites.8 These other metabolites included: 1-hydroxy, 12-hydroxy, 14-hydroxy, 49-
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hydroxy, 2 hydroxy-piperidine, EVR, 16-O-desmethyl, 16,39-O-didesmethyl, 16,27-Odidesmethyl, and 27,39-O-didesmethyl EVR. Total metabolites ranged from 50% to 100% of EVR concentrations (0.1 to 0.25 mg/L). Clinical relevance of these metabolites awaits further study. EVR TDM for transplant was assessed by a consensus conference under the auspice of the International Association for TDM and Clinical Toxicology (IATDMCT), followed by published
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ACCEPTED MANUSCRIPT reports. 9-11 From the conference, Billaud detailed TDM issues included CNI minimization for lowering nephrotoxicity, EVR for treating malignancy and use in preventing rejection and late graft dysfunction. 4 CNI minimization would include conversion from CNI to EVR and de novo dosing for high risk renal failure patients. However, a recent study in converting CsA to EVR
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with mycophenolate sodium showed limited tolerability and efficacy.12 Hesselink’s
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recommended the practice of EVR TDM including: monitoring pre-dose whole-blood
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concentrations in early transplant with a proposed trough concentration range of 4-8 ng/mL, and in late period, 3-4 to 6 ng/mL for follow-up and/or for checking toxicity, suggested to be based
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on a “moderate” degree of evidence. 13 Generally, in combination therapy with cyclosporine,
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therapeutic trough concentration range is 3– 8 ng/mL, based on LC-MS/MS assay of the parent drug. Currently available EVR assays might be used to maintain efficacy and minimize toxicity,
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due to the large inter-patient and possibly intra-patient variabilities, for drug-drug interactions
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and non-compliance. Holt addressed analytical methodology development, accompanying issues of calibrations, changing prescribing practice, simultaneous monitoring of multi-drugs, shorter
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analysis time and reproducibility. 14 EVR monitoring by immunoassays 3,15-22 and
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chromatography in combination with mass spectrometry are, according to Holt ,14 inherently problematic.6,14,19,22,23
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This study of QMS EVR TDM evaluated assay performance – linearity, precision, accuracy and methods comparison, performed by four analyzers – Indiko, DxC, AU 680 and AU5800 in three sequential periods. Period I initially evaluated the QMS EVR assay performance on the Indiko and DxC analyzers. De-identified samples were obtained from three sources: Wake Forest, ARUP Laboratories and University of Colorado. This was followed by periods II and III, as a result of two automation updates in our chemistry core laboratory during the past three and half
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ACCEPTED MANUSCRIPT years (2012 to mid-2015).24 Period II closely coincided with the first update involving the placement of two AU680 analyzers at the beginning of 2013. It also included comparison of QMS to LC-MS/MS performed with de-identified patient samples from ARUP. Period III evaluation commenced in 2015 when AU5800 analyzers replacing two AU680 analyzers.
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Materials and Methods
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QMS EVR assay materials for periods I, II and III evaluations were obtained from Thermo Scientific (Fremont, CA). Assay parameters for this user-defined test were programmed
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according to the manufacturer’s recommendation. 25
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Materials for LC-MS/MS assay
To support the method comparison evaluation, EVR and zinc sulfate monohydrate was
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purchased from Mass Supply Company (Highlands Ranch, CO). EVR-d4 was obtained from
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Toronto Research Chemicals (Toronto, Canada). HPLC grade methanol was purchased from
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VWR Scientific (Radnor, PA). Calibrator materials were obtained from ChromSystems (6PLUS1 Multilevel Whole Blood Calibrator Set, Gräfelfing, Germany) and quality control
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materials were purchased from UTAK Laboratories (Whole Blood Toxicology Control Level
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1&2, Valencia, CA).
Methods Validations and Comparisons QMS assay QMS EVR is a turbidimetric immunoassay. Sample preparation was initiated by mixing 300 L of sample (patient whole blood) with 350 L of methanol and 50 L of a precipitation reagent. After vortexing for 35 seconds and centrifugation for 8 minutes at 13,000 xg, 350 L of the
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ACCEPTED MANUSCRIPT supernatant was transferred to sample cups. Drug in the supernatant and drug coated on microparticle compete for the limited number of antibody binding sites. If EVR is absent in the sample supernatant, EVR-coated microparticles are agglutinated in the presence of antibody reagent. Agglutination is measured photometrically. If EVR is present, agglutination is partially
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inhibited depending on EVR concentration. Thus, the agglutination rate is inversely proportional
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to EVR concentration. Calibrators range was from 0, 1.5, 3.0, 6.0, 12.0 and 20 ng/mL. Precision
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and comparison studies were performed with de-identified patient samples. In establishing accuracy, a de-identified patient sample with a high EVR concentration was diluted with whole
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blood hemolysate and assayed five times. For Period 1, samples from renal transplant patient
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samples from were re-analyzed the following time periods post collections: Wake Forest, 1 to 2 weeks., ARUP, 2 to 3 months., and University of Colorado, 2 to 6 months., for Period II, ARUP
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samples within 2 to 3 months, and for Period III, from Wake Forest, within 1 to 2 weeks. These
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samples underwent 2 to 4 freeze and thaw cycles.
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Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay The EVR LC-MS/MS method was performed according to the recent publication by Johnson-
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Davis et al. 2015. 6 Data analysis was performed using Analyst® software and the concentration
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of EVR was calculated by applying the ratio of the peak areas of EVR to the corresponding internal standard. The peak area ratio from a patient sample was then compared to a calibration curve formed by a series of calibrators of known concentrations within the same matrix. Statistical analysis (Deming Regression Analysis and Bland-Altman Plot) was performed by the Biostatistics Department of Wake Forest School of Medicine. Results and Discussion
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ACCEPTED MANUSCRIPT The study may be readily divided into three sequential periods: Period I 2012-2013 - QMS assays comparison with DxC, AU680 and Indiko using deidentified samples from Wake Forest, ARUP and University of Colorado Period II 2013-2014 - comparison of LC-MS/MS with AU 680 and Indiko using de-
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identified samples from ARUP only
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Period III 2015-2016 - comparison of AU680 and AU5800 analyzers using de-identified
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samples from Wake Forest only.
Periods I and III studies of QMS assays by Indiko, DXc, AU680, and AU5822
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Linearity data of Periods I and III studies of Indiko, DxC, AU680, and AU5800, summarized in
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Table 1, showed analytical measuring ranges of 0.51 to 20 ng/mL with slopes ranging from 1.002 to 1.025. The lower limit of analytical measurement range was established as the limit of
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detection, determined by repeated dilution of the low calibrator, 1.5 ng/mL with imprecision
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<20%. Limit of quantitation for patient EVR monitoring was set at the low calibrator of 1.5 ng/mL. Calibration stabilities ranged from 1 day for DxC, different from the manufacturer’s
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claim, to 5 days for Indiko, AU680 and AU5800. Accuracy was established by dilution of a de-
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identified patient sample with high EVR concentration, and by comparison studies of Indiko, AU680 and LC-MS/MS. A de-idenified patient sample with EVR concentration of 13.6 ng/mL,
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diluted with whole blood hemolysate to low concentration of 1.8 ng/mL, and these were assayed five times. The corresponding concentrations, recoveries and CVs of these two samples sets analyzed by Indiko, DxC and AU 680 ranged from: 11.99 – 14.21 and 1.81 – 1.94 ng/mL., 98 101.56 and 93 - 115%., and 3.75 - 4.5 and 18.50 – 20.85 %. Day-to-day precision studies, shown in Table IB, showed CVs of 2.6 to 8.9% for the low, medium and high controls. Period I comparison studies of the three analyzers, shown in Table 2, included 107 de-identified
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ACCEPTED MANUSCRIPT transplant samples with concentrations ranging from <1.5 to 13.6 ng/mL. Deming regression showed the 5 slopes ranging from 1.000 to 1.076, acceptable intercepts and correlations, Sy/x ranging from 0.49 to 0.78. Period III, performed in early 2015 after the second automation update, compared AU 680 to AU5822 analyzers, showing slope of 1.007. This later comparison
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Period II study of LC-MS/MS and QMS assays comparison
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was performed with a limited number of samples.
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Period II study included 104 de-identified EVR patient samples obtained from ARUP. The samples were analyzed by LC-MS/MS, and later re-analyzed by QMS EVR assays using Indiko
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and AU680 in our lab. Performance data and statistical analyses using Deming regression and
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Bland-Altman plots are shown in Figures 1a-f and Table 3. Comparison studies between LCMS/MS vs AU680, LCMSMS vs Indiko, and AU 680 vs Indiko showed slopes of 1.046, 1.086
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and 1.035 respectively., and the corresponding mean differences, as shown by the Bland-Altman
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plots, were: 0.05 (95% CI -1.83 to 1.84), -0.45 (95% CI -2.74 to 1.83) and -0.47 (95% CI -1.63 to 0.85). Mean concentrations for LC-MS/MS, AU680 and Indiko were 5.403, 5.393, 5.849
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ng/mL respectively and Sy/x ranged from 0.53 to 0.74 ng/mL. Indiko results showed a positive
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bias, with the mean concentration of 5.849. The differences of immunoassay platforms and LCMS/MS, the time elapsed between the two assays, and sample handling might have contributed
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to the data scattering, as shown by Deming regression and Bland-Altman plots. Within the period II evaluation, 42 de-identified samples from 11 renal transplant patients were monitored over various time periods, as shown by Supplemental Table 3. EVR concentrations of renal transplant patients were 88% within the recommended range of 3-4 to 6-8 ng/mL as suggested by Hesselink.13
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ACCEPTED MANUSCRIPT QMS EVR monitoring proficiency performance, as shown by Supplemental Table 4, was evaluated by our enrollment with the College of American Pathologists (CAP) Survey, using AU680 for 2013-2014, and AU5800 for 2015 - 2016. Standard Deviation Index (SDIs) and the means ranged from:-1.7, Wake Forest (WF) and QMS means of 11.1 and 11.98., to +1.8, WF
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and QMS means of 6.0 and 5.05 respectively. These differences would most likely not have
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changed EVR TDM.
For 2014-2016 CAP EVR annual surveys, there were two series (EV-A and EV-B) with three
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concentrations – low, medium and high. For the low concentration samples, the reported ranges (ng/mL) of LC-MS/MS assays, PETINTA assays, and difference ranges of the two assays were:
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2.0 to 2.2, non-detected (ND) to 2.0, and ND to 2.0 respectively., for medium concentrations,
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7.9 to 8.5, 5.1 to 5.5, and 2.4 to 3.1., and for high concentrations, 16.8 to 21.6, 12.4 to 14.4 and 4.3 to 7.1 respectively. In assessing EVR TDM, the difference range of medium concentrations
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of 2.4 to 3.1 would be of interest, and might be explained by the formulation of the assay
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calibrators at about 70% of the gravimetrically-spiked samples as explained in a later section.
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Comparison of this study with previous publications In two previous publications comparing immunoassays as shown by Supplemental Table 1,
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Buthiau et al. compared FPIA/Innofluor to QMS Indiko, showing a slope of 0.81, possibly due to different immunoassay chemistry. 19 However, Mori et al. compared FPIA/Innofluor to Nanopia/QMS, showing an acceptable slope of 0.966. 22 For comparisons of LC-MS/MS assays to QMS immunoassays, findings from previous five publications, as shown by Supplemental Table 2, showed slopes ranging from 0.8 to 1.277. Hopfer compared QMS EVR performed on Architect (Abbott) analyzers to LC-MS/MS for monitoring 250 samples from 169 renal, heart, lung, and liver transplant patients. 20 Mean 10
ACCEPTED MANUSCRIPT concentrations for LC-MS/MS and QMS were 4.8 and 6.3 ng/mL respectively. Above therapeutic concentrations by QMS accounted for 69% of samples which were shown to be therapeutic by LC-MS/MS. This lack of agreement resulted in the authors’ suggestion that these assays did not yield interchangeable EVR concentrations. Shu et al compared LC-MS/MS to
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EVR QMS assay by Vitros using 34 samples.21 This later study showed a “high” slope of 1.271,
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but the authors concluded that the assay was “reliable and reproducible”.18 The results of these
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two studies are different from findings of two other studies by Dasgupta et al.3 and Buthaiau.19 Dasgupta ‘s compared LC-MS/MS to QMS assay by using Hitachi 917, showing a slope of 1.11. Buthaiau compared the QMS EVR assay using Indiko to UPLC-MS/MS and an
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3
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Innofluor/FPIA assay (Seradyn Inc) for 120 samples from 74 transplant patients (35 kidney, 30 liver and 9 heart).19 The study showed QMS EVR on the Indiko was reliable with acceptable
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accuracy when compared to UPLC-MS/MS, UPLC-MS/MS to QMS by Indiko, showing a slope
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of 0.99.while the Innofluor assay (FPIA, Abbott, recently discontinued) over-recovered by about 21%,19 similar to a 24% higher measurement observed in an earlier published study. 22
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These results are similar to our Period II comparison study of using Indiko and AU680 analyzers,
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and LC-MS/MS showed more comparable and acceptable slopes ranging from 1.046 to 1.086. The difference in slopes might be attributable to multiple factors – sample types, and calibration
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and variability of the study methods including LC-MS/MS. This was one of the findings from the long-term cross-validation Zortracker Study,26 as well as one of the conclusions of Holt’s presentation at the 2014 EVR Consensus Conference. 14 Further, the differences between these studies might be partially attributed to QMS EVR assay calibration. As described by the manufacturer in the QMS EVR package insert, “calibrators and controls are prepared gravimetrically by adding EVR to human whole blood. According to the
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ACCEPTED MANUSCRIPT manufacturer, to minimize bias between QMS and LC-MS/MS results, value assignment of calibrators and controls were based on a set of clinical trough EVR patient samples and would result in “under-recovery “at 70% of the gravimetrically-spiked samples.13 This might explain the CAP EVR surveys summarized in Supplemental Table 4 showing negative basis for the
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QMS assay group with testing EVR-spiked samples rather than patient-derived samples.
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According to the manufacturer, EVR metabolites cross-reactivities ranged from 2 – 63%.25
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However, a recent study showed the addition of two specific EVR metabolites did not affect the Zortracker survey results. 26 In assessing the EVR TDM performance from multi-centers,
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Schniedewind et al. sent EVR samples from a prospective clinical trial to participating
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laboratories: LC-MS/MS (n=6 to13) and QMS (n = 4-8 ) assays.26 In order to test for interference, two metabolites, 39-O-desmethyl and 46-hydroxy EVR were added to two
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individual pools. The analyses of these two sample pool samples (Feb. 2013 to Feb. 2014),
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showed comparable means (ng/mL) and ranges for 107 LC-MS/MS and 77 QMS assays samples as follows: 5.31 and 2.9 – 7.8 ng/mL, and 5.20 and 4.0 – 6.8 ng/mL respectively. Thus, the
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authors suggested that the two added metabolites did not interfere with the assays.
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Shortly after this publication, the influence of laboratory practices on immunosuppressant
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monitoring variability was investigated using data generated by an immunosuppressants survey sponsored by Novartis and under the auspice of IATDMCT.27 From the 2013 survey of 76 participating laboratories in 14 countries, the variability was due to differences in lab procedures, lack of standardization in pre-analytics, use of inappropriate reference materials, and noncompliance to accepted international guidelines. This might be improved by standardized operational procedures and personnel proficiency. Limitations 12
ACCEPTED MANUSCRIPT For the period I and II studies, de-identified samples and their LC-MS/MS concentrations were mostly provided by outside laboratories, and our QMS measurements were “delayed” for several weeks relative to the LC-MS/MS measurements. This delay, with time period comparable to that of performing CAP survey, did not contribute to significant variability. As shown by CAP EVR
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surveys, LC-MS/MS and EVR QMS assays of “spiked “ survey samples showed the “expected “
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significant differences.
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Conclusions: EVR may be monitored by the QMS assay using four analyzers - Indiko, DxC, AU680 and AU5822 with adequate limit of detection and acceptable precision, showing high
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correlation of the four analyzers. Over the past 3.5 years, we have found these four analyzers
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offered TDM EVR monitoring suitable for renal and liver transplant recipients.
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ACCEPTED MANUSCRIPT Acknowledgements: This study was supported by an instrument/reagent evaluation grant from Thermo Fisher Scientific. The co-authors greatly appreciated the provisions of left-over EVR samples and
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guidance by colleagues from University of Colorado.
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ACCEPTED MANUSCRIPT References: 1. Kirchner GI, Meier-Wiedenbach I, Manns MP. Clinical pharmacokinetics of everolimus. Clin Pharmacokinetic., 2004;43:83-95. 2. Kuhn B, Jacobsen W, Christians U, Benet LZ, Kollman PA. Metabolism of Sirolimus and
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its Derivative Everolimus by Cytochrome P450 3A4: Insights from Docking, Molecular
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Dynamics, and Quantum Chemical Calculations. J Med Chem;2001:44:2027-34.
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3. Dasgupta A, Davis B, Chow L. Evaluation of QMS Everolimus Assay using Hitachi 917 analyzer: Comparison with Liquid Chromatography/Mass Spectrometry. Ther Drug
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Monit. 2011;33:149-54.
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4. Billaud EM. Clinical pharmacology of Everolimus with focus on implication on Therapeutic Drug Monitoring. Accessed: January 10, 2016.
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https://www.iatdmct.org/resources/presentations/426-rsc-pres-isd-20140911.html.
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5. Gabardi S, Baroletti SA. Everolimus: a proliferation signal inhibitor with clinical
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applications in organ transplantation, oncology, and cardiology. Pharmacotherapy.
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6. Johnson-Davis KL. Juenke JM, Thomas RL, Bradshaw T. Everolimus method comparison between Waters MassTrakTM immunosuppressants XE(IUO) kit and an in-
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house laboratory developed LC-MS/MS method in renal transplant patients. Ann Clin Lab Sci. 2015;45:27-31. 7. Picard N, Bergan S, Marquet P, van Gelder T, Wallemacq P, Hesselink DA, Haufroid V. Pharmacogenetic biomarkers predictive of the pharmacokinetics and pharmacodynamics of immunosuppressive drugs. Ther Drug Monit. 2016;38(S1):S57-S69.
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ACCEPTED MANUSCRIPT 8. Strom T, Haschke M, Zhang YL, et al. Identification of Everolimus metabolite Patterns in Trough Blood Samples of Kidney Transplant Patients. Ther Drug Monit. 2007;29:5929. 9. ITADMCT – Immunosuppressive drugs consensus conference presentations. September,
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https://iatdmct.org/resources/presentations/426-rsc-pres-isd-20140911.html
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10. Shipkova M, Hessleink DA, Holt DW, Billaud EM, van Gelder T, Kunickt PK, et al. Therapeutic Drug Monitoring of Everolimus: A Consensus Report. Ther Drug Monit.
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11. Seger C, Shipkova M, Christians U, Billaud E, Wang P, Holt D, Brunet M, Kunicki PK, Pawiński T, Langman LJ, Marquet P, Oellerich M, Wieland E, Wallemacq P.
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Assuring the Proper Analytical Performance of Measurement Procedures for
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12. Felipe C, Oliveira N, Hannun P, de Paula MI, Tedisco-Silva H, Medina-Pestana JO. Pharmacokinetics and long-term safety and tolerability of Everolimus in renal transplant
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recipients converted from cyclosporine. Ther Drug Monit. 2016;38:64-72. 13. Hesselink DA. Practice of TDM of Everolimus: An overview: Accessed: January 10, 2016. https://www.iatdmct.org/resources/presentations/426-rsc-pres-isd-20140911.html. 14. Holt DW. Analytical aspects of the determination of Everolimus concentrations. Accessed: January 10, 2016. https://www.iatdmct.org/resources/presentations/426-rscpres-isd-20140911.html.
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ACCEPTED MANUSCRIPT 15. Wong SHY, Johnson-Davis K, Garrison K Evaluation of Everolimus QMS assay by using Thermo Scientific Indiko, Beckman DxC, and AU680 analyzers. Clin Chem. 2013;59:A135. 16. Wong C, Cheng D, Naqvi H, Yokobata. QMS EVR assay for Beckman Coulter AU480,
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AU680, AU5800 Clinical Chemistry Analyzers. Clin Chem. 2014;60:S150.Baldelli S,
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Crippa A, Gabrieli R, et al. Comparison of the Innofluor Certican assay with HPLC-UV
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for the determination of Everolimus concentrations in heart transplantation. Clin Biochem. 2006;39:1152-1159
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17. Baldelli S, Crippa A, Gabrieli R, et al. Comparison of the Innofluor Certican assay with
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HPLC-UV for the determination of Everolimus concentrations in heart transplantation.
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18. Wong C, Cheng D, Thao A, Ye L. QMS Tacrolimus assay for Beckman Coulter AU480,
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AU680, AU5800 Clinical Chemistry Analyzers. Clin Chem. 2014;60:S145. 19. Buthiau D, Bargnoux A, Badiou S, Sutra T, Dupuy A, Pageaux G, Mourad G, Cristol J.
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performance liquid chromatography-tandem mass spectrometry method. Ther Drug Monit. 2015;37:275-278.
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20. Hoffer E, Kurnik D, Efrati E, Scherb I, Karasik M, Ring G, Bentur Y. Comparison of Everolimus QMS immunoassay on Architect ci4100 and liquid chromatography/mass spectrometry: Lack of agreement in organ-transplanted patients. Ther Drug Monit. 2015;37:214-219.
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ACCEPTED MANUSCRIPT 21. Shu I, Wright AM, Chandler WL, Bernard DW, Wang P. Analytical performance of QMS Everolimus assay on Ortho Vitros 5,1 FS Fusion analyzer: Measuring EVR trough levels for solid organ transplant recipients. Ther Drug Monit. 2014;36:264-268. 22. Mori K, Kimura S, Matsui M, Suehisa E, Hidaka Y, Fufushima N. Latex-enhanced
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turbidimetric immunoassay for Everolimus in whole blood using the Nanopia TDM EVR
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assay with the JCA-BM6010 automatic analyzer. Ther Drug Monit. 2014;36:677-680.
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23. McMillin GA, Johnson-Davis K, Dasgupta A. Analytical performance of a new liquid chromatography/tandem mass spectrometric method for determination of Everolimus
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concentrations in whole blood. Ther Drug Monit. 2012;34:222-226
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24. QM Zimmerman MK, Friesen LR, Nice A, Vollmer PA, Dockery EA, Rankin JD, Zmuda K, Wong SHY. Multi-center evaluation of analytical performance of the Beckman Coulter
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AU5822 chemistry analyzer. Clin Biochem.2015;48:881-885.
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25. Everolimus (Package insert). Fremont, CA Thermo Fisher, 2010. 26. Schniedewind B, Niederlechner S, Galinkin JL, Johnson-Davis KL, Christians U, Meyer
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EJ. Long-term cross-validation of Everolimus therapeutic drug monitoring assays: The
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Zortracker study. Ther Drug Monit. 2015;37:296-303. 27. Christians U, Vinks AA, Langman LKJ, Clarke W, Wallemacq P, van Gelder T, Renjen
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V, Marquet P, Meyer EJ. Impact of Laboratory practices on interlaboratory variability in Therapeutic Drug Monitoring of immunosuppressive drugs. Ther Drug Monit. 2015;37:718-724.
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ACCEPTED MANUSCRIPT Table 1 - Period I and III QMS EVR assay performance Linearity of QMS EVR assay by Indiko, AU680, DxC and AU5800 Analytical
Slope
R2
Intercept
measuring
ng/mL
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ranges, ng/mL
Indiko
0.53-20
1.014
0.121
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AU680
0.51 – 19
1.025
0.105
0.998
0.51-20
1.023
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DxC
0.195
0.76 – 20.0
0.674
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1.002
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Period III AU5822
0.997
19
0.998
0.968
ACCEPTED MANUSCRIPT Table 1B: Period I and III Day-to-Day Precision studies for Indiko, DxC, AU680 and AU5800(n
= 20 and study duration = 2 weeks). Sample
Mean conc.(ng/mL)
CV, %
Analyzer Period I
DxC
Medium
7.81
High
15.25
3.1
Low
4.72
3.6
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5.4
7.70
4.5
15.56
2.6
4.20
3.1
7.80
3.4
14.90
3.5
Low
4.10
8.7
Medium
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8.9
High
14.80
4.4
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AU5822
6.8
Period III
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AU680
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Intercepts, ng/mL
DxC vs. Indiko
107
Period I 1.073 (1.019 to 1.127)
AU680 vs. Indiko
107
1.076 (1.022 to 1.134)
DxC vs. AU680
107
1.000 (0.961 to 1.038)
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Period III 1.007 (0.957 to 1.057)
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0.434 (0.192 to 0.843)
0.945
0.71
0.462 (0.221 to 0.814)
0.962
0.78
-0.053 (-0.262 to 0.154)
0.981
0.49
-0.265 (-0.622 to 0.038)
0.980
0.53
-0.802 (-1.012 to -0.593)
0.952
0.61
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AU680 vs AU5822
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Sy/x ng/mL
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Slopes
Period II 1.035 (0.983 to 1.087)
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Table 2 – Correlation data of Immunoassays analyzers comparison with de-identified transplant samples: for Period I, from ARUP, University of Colorado and Wake Forest., for Period II from ARUP., and for Period III from Wake Forest.
ACCEPTED MANUSCRIPT Table 3: Summaries of comparison of LC-MS/MS with QMS EVR assays of de-identified samples from ARUP for the Period II study
Intercepts ng/mL
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n
Mean LCMS/MS ng/mL
Mean QMS AU680 ng/mL
Mean QMS Indiko ng/mL
Sy/x ng/mL
LC-MS/MS vs AU680
1.046 (0.981 to 1.111)
-0.256 (-0.561 to 0.042)
0.945
104
5.403
5.393
--
0.74
LC-MS/MS vs Indiko
1.086 (1.025 to 1.147)
-0.019 (-0.332 to 0.293)
0.924
104
5.403
--
5.849
0.71
AU680 vs Indiko
1.035 (0.983 to 1.087)
-0.265 (-0.622 to 0.038)
0.980
104
5.393
5.849
0.53
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Slopes
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Slope
Intercept, ng/mL
DxC vs. Indiko AU680 vs. Indiko DxC vs. AU680
107 107 107
This study Period I 1.073 1.076 1.000
0.434 0.462 -0.053
0.945 0.962 0.981
DxC vs AU680
104
Period II 1.035
-0.265
0.980
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Period III 1.007
-0.802
0.952
Other publications 0.81
-0.09
0.94
0.946
NA
0942
120
91
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Innofluor/FPIA vs QMS Indiko, Buthaiau (19) Innofluor/FPIA vs Nanopia/QMS Mori (22)
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AU680 vs AU5822
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Analyzers, author (reference)
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n
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Supplemental Table 1 – Correlation data of Immunoassays analyzers comparison of Periods I, II and III of this study with de-identified transplant samples (refer to table 2 for sample sources) and other recent publications
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NA, Not available
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ACCEPTED MANUSCRIPT Supplemental Table 2: Summaries of comparison of LC-MS/MS with QMS EVR from Period II of this study and other studies of immunoassays (NA – Not available)
n
Mean LCMSMS
Mean QMS
Comments
Period II of this study 1.046
0.945
104
5.403
5.393
Refer to the results and discussion of this paper
1.086
0.924
104
5.403
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5.849
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LC-MS/MS vs AU680
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90
NA
NA
0.95
120
5.3
5.2
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Wong et al This study
Slopes
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Method comparison
0.8
0.66
250
4.8
6.3
LC-MS/MS vs Nanopia/QMS
0.960
0.946
91
6.41
6.47
LC-MS/MS vs QMS(Vitros)
1.271
0.880
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NA
NA
LC-MS/MS vs QMS (Vitros)
1.277
0.912
8 (liver only)
NA
NA
LC-MS/MS vs Indiko
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References
Other publications
Buthaiau et al, 2015 (19)
UPLC-MS/MS vs QMS Indiko
0.99
Hopfer et al. 2015 (20)
LC-MS/MS vs QMS Architect
Mori et al 2014 (22) Shu et al 2014 (21)
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0.92
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1.11
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LC-MS/MS vs QMS Hitachi
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Dasgupta et al (3)
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Adequate sensitivities, specificity and analytical measurement range QMS - Reliable and reproducible performance QMS - Significant positive bias – not used interchangeably Nanopia/QMS Suitable for routine use QMSSatisfactory analytical performance QMS Satisfactory analytical performance
ACCEPTED MANUSCRIPT Supplemental Table 3 - Period III EVR concentrations by using AU 5822 for 42 de-identified samples from 11 renal transplant patients
28/6 21/6
69/5 34/5
51/5
13/6 4/3
20/6 6/2*
26/6 7/<2*
34/4 8/<2*
27/4 16/5 35/5 49/4
55/4 28/5
** Above therapeutic
51/5 28/7
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14/5 7/6
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* Subtherapeutic
7/5 1/6 55/5 7/8 3/3
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0/5 0/8 0/7 0/9** 0/3 0/3 0/4 0/4 0/4 0/10** 0/5
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1 2 3 4 5 6 7 8 9 10 11
Days from the first sample/conc. ng/mL
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Patient numbers
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61/5 32/7
75/4
ACCEPTED MANUSCRIPT Supplemental Table 4 – CAP EVR survey results from Wake Forest (WF) and peer groups from 2013 to 2016 CAP surveys for QMS EVR PETINA (PET)** and LC-MS/MS (LCMS). EVR concentration, ng/mL
LC-MS
EV-A
1 2 3
12.0 <2.0 5.0
12 1.5 5.2
17.6 2.1 8.4
<2.0 5.0 12.0
SDI *** -1.2 -0.4
EV-B
4 5 6
<2.0 5.0 12.0
1.1 5.2 11.3
2.1 8.2 17.7
4.9 11.1 <2.0
-1.5 -1.7 ***
1 2 3
5.0 13.0 3.0
EV-B
4 5 6
15.0 2.0 6.0
1.1 1.2 1.8
5.52 11.98 ***
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EV-A
SDI -0.1 -1.2 1.4
PET Mean 5.1 13.7 1.6
SD 0.73 0.58 1.02
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2015 WF
LC-MS Mean 8.18 20.16 2.14
SD 1.21 2.93 0.44
13.88 1.14 5.05
1.02 0.10 0.54
20.98 2.18 8.18
3.06 0.34 1.11
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Sample No.
PET Mean *** 5.5 12.44
SD *** 0.46 0.99
LC-MS Mean 2.0 7.9 16.76
SD 1.29 1.12 2.34
0.41 0.53 ***
8.5 17.77 2.17
1.04 2.05 0.34
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PET
2014 WF
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2013** WF
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CAP
Sample No.
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CAP
2016 WF 16.0 <2.0 6.0
SDI 1.3 1.0
PET Mean 14.42 1.62 5.49
SD 1.24 0.71 0.53
LC-MS Mean 20.57 2.22 8.51
SD 2.34 0.36 1.23
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* Standard Deviations (SDs) of 2013 surveys were no longer available as a result of the normal practice of keeping survey results for only two recent years
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**QMS results were grouped under PETINA(PET) by CAP *** No mean and SD reported for results of less than 10 reporting labs SDI – Standard deviation Index = (WF EVR concentration – EVR mean concentration)/EVR SD
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ACCEPTED MANUSCRIPT Figure Legends Figure 1 Deming Regression and the Bland-Altman bias plots for comparisons of QMS EVR assays of 104 EVR patient samples from ARUP. The inner blue line of the Bland-Altman plots represents the mean difference of the two assays, and outer blue lines are the 95% CI of the mean difference.
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Figures 1a and 1b show the Deming Regression and the Bland-Altman bias plot for comparison of EVR assays by LC-MS/MS and QMS Indiko.
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Figures 1c and 1d show the Deming Regression and the Bland-Altman bias plot for comparison of EVR assays by LC-MS/MS and QMS AU680.
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Figures 1e and 1f show the Deming Regression and the Bland-Altman bias plot for comparison of QMS EVR assays using AU680 and Indiko.
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Figure 1a
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Figure 1b
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Figure 1c
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Figure 1d
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Figure 1e
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Figure 1f
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v v
Steven HY Wong, Kamisha L Johnson-Davis, Krista Garrison, Joe D Rankin, Chro S Muhammad PII: DOI: Reference:
S0009-9120(16)30264-8 doi: 10.1016/j.clinbiochem.2016.12.003 CLB 9444
To appear in:
Clinical Biochemistry
Received date: Revised date: Accepted date:
12 September 2016 7 December 2016 9 December 2016
Please cite this article as: Steven HY Wong, Kamisha L Johnson-Davis, Krista Garrison, Joe D Rankin, Chro S Muhammad , Everolimus TDM using Thermo Fisher QMS immunoassay on Indiko, Beckman DxC, AU680, and AU5800 analyzers. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Clb(2016), doi: 10.1016/j.clinbiochem.2016.12.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Everolimus TDM using Thermo Fisher QMS immunoassay on
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Indiko, Beckman DxC, AU680, and AU5800 analyzers
Steven HY Wong1,2*, Kamisha L Johnson-Davis 3,4, Krista Garrison1,
Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT
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3
Wake Forest School of Medicine, Winston-Salem, NC
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2
Wake Forest Baptist Health, Winston-Salem, NC,
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1
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Joe D Rankin 1, and Chro S Muhammad 2,5,6
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Sulaimania School of Medicine, Sulaimania, Kurdistan,
Kurdistan Board of Medical Science (KBMS), Kurdistan
*Corresponding author – Steven H. Wong, Ph.D., Department of Pathology, Wake Forest School of Medicine, One Medical Center Boulevard, Winston-Salem, NC 27157, USA. Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Objectives: Everolimus (EVR), a mTOR inhibitor immunosuppressant approved for renal, liver and cardiac transplants. This study established the appropriate TDM performances of the Thermo Scientific QMS EVR Assay by using the Beckman DxC, followed by comparison to the Thermo
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Scientific Indiko and a previously published LC-MS/MS assay., and Beckman AU680 and
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AU5800 analyzers.
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Design and Methods: The study initially established acceptable linearity, precision and accuracy of the QMS EVR turbidimetric assay. Sample preparation was initiated by mixing patient whole
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blood with methanol and a precipitation reagent. Supernatant was transferred to sample cups.
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Drug in the supernatant and drug coated on microparticle compete for the limited number of antibody binding sites. If EVR is absent in the sample supernatant, EVR coated microparticles
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are agglutinated in the presence of antibody reagent. If EVR is present, agglutination is partially
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inhibited, depending on EVR concentration. Calibrators range was from 1.5 to 20 ng/mL. Comparison studies data were analyzed by Deming Regression and Bland-Altman plots.
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Results: Precision studies showed the following mean concentrations 4.00 – 4.72, 7.70 – 8.20
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and 14.80 – 15.56 ng/mL, and CVs of 3.1 – 8.7%, 3.4 – 8.9% and 2.6 – 4.4 % respectively. Comparison of analyses of 107 de-identified transplant samples by three analyzers- Indiko, DxC
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and AU680 showed: EVR concentrations from <1.5 to 13.6 ng/mL, slopes 1.000 to 1.076, intercepts -0.053 to 0.462, and R 0.945 to 0.981. Another series of comparison studies (n=104) of Indiko and AU680 with LC-MS/MS showed the following: slopes 1.035 to 1.086, intercepts -.019 to -0.265, and R 0.924 to 0.980. 2013-2016 CAP survey results were acceptable.
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ACCEPTED MANUSCRIPT Conclusions: Based on the experience of the past 3.5 years, Thermo Scientific QMS EVR Immunoassay using four different analyzers offered adequate limit of detection and acceptable
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accuracy and precision, suitable for monitoring renal and liver transplant recipients.
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Keywords
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Everolimus TDM, autoanalyzers, immunoassay, LC-MS/MS, performance evaluation, multi-center study, survey results
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ACCEPTED MANUSCRIPT Introduction: Everolimus (EVR) is a mTOR inhibitor immunosuppressant with FDA approvals for renal and liver transplants.1-4 EVR is often used in combination with calcineurin inhibitors (CNI) such as cyclosporine and tacrolimus (FK-506). It has also been used for cardiac transplant. The marketing names for EVR are: Zortress in U.S., Certican for Europe and other countries, and
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Afinitor for cancer treatment. EVR can also be used for treating cancers, advanced breast cancer,
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and the orphan disease tuberous sclerosis complex.5
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subependymal giant cell astrocytoma, neuroendocrine tumors of pancreatic origin, renal cancers
EVR is rapidly absorbed, with blood concentration peaking between 1.3-1.8 hr, half- life ranging
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18-35 hours, and bioavailability ranging from 5-26%. With twice daily dosing, steady state might
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be attained within 7 days.1,4 Age, weight or sex do not affect pharmacokinetics in adults. EVR is metabolized by CYP 3A4, 3A5 and 2C8. EVR pharmacogenetics was also recently and
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extensively reviewed by Johnson-Davis,6 and Picard.7 Further metabolism study employed
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quantum chemical calculations and molecular simulations which predicted region-specific hydroxylations and O-dealkylations.2 Using a combination of high-resolution TOF MS,
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hydrogen/deuterium exchange and LC-ion trap mass spectrometry, key fragmentation pathways
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of 24-hydroxy EVR were characterized, using 128 patient samples, leading to identification of other metabolites.8 These other metabolites included: 1-hydroxy, 12-hydroxy, 14-hydroxy, 49-
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hydroxy, 2 hydroxy-piperidine, EVR, 16-O-desmethyl, 16,39-O-didesmethyl, 16,27-Odidesmethyl, and 27,39-O-didesmethyl EVR. Total metabolites ranged from 50% to 100% of EVR concentrations (0.1 to 0.25 mg/L). Clinical relevance of these metabolites awaits further study. EVR TDM for transplant was assessed by a consensus conference under the auspice of the International Association for TDM and Clinical Toxicology (IATDMCT), followed by published
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ACCEPTED MANUSCRIPT reports. 9-11 From the conference, Billaud detailed TDM issues included CNI minimization for lowering nephrotoxicity, EVR for treating malignancy and use in preventing rejection and late graft dysfunction. 4 CNI minimization would include conversion from CNI to EVR and de novo dosing for high risk renal failure patients. However, a recent study in converting CsA to EVR
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with mycophenolate sodium showed limited tolerability and efficacy.12 Hesselink’s
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recommended the practice of EVR TDM including: monitoring pre-dose whole-blood
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concentrations in early transplant with a proposed trough concentration range of 4-8 ng/mL, and in late period, 3-4 to 6 ng/mL for follow-up and/or for checking toxicity, suggested to be based
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on a “moderate” degree of evidence. 13 Generally, in combination therapy with cyclosporine,
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therapeutic trough concentration range is 3– 8 ng/mL, based on LC-MS/MS assay of the parent drug. Currently available EVR assays might be used to maintain efficacy and minimize toxicity,
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due to the large inter-patient and possibly intra-patient variabilities, for drug-drug interactions
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and non-compliance. Holt addressed analytical methodology development, accompanying issues of calibrations, changing prescribing practice, simultaneous monitoring of multi-drugs, shorter
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analysis time and reproducibility. 14 EVR monitoring by immunoassays 3,15-22 and
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chromatography in combination with mass spectrometry are, according to Holt ,14 inherently problematic.6,14,19,22,23
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This study of QMS EVR TDM evaluated assay performance – linearity, precision, accuracy and methods comparison, performed by four analyzers – Indiko, DxC, AU 680 and AU5800 in three sequential periods. Period I initially evaluated the QMS EVR assay performance on the Indiko and DxC analyzers. De-identified samples were obtained from three sources: Wake Forest, ARUP Laboratories and University of Colorado. This was followed by periods II and III, as a result of two automation updates in our chemistry core laboratory during the past three and half
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ACCEPTED MANUSCRIPT years (2012 to mid-2015).24 Period II closely coincided with the first update involving the placement of two AU680 analyzers at the beginning of 2013. It also included comparison of QMS to LC-MS/MS performed with de-identified patient samples from ARUP. Period III evaluation commenced in 2015 when AU5800 analyzers replacing two AU680 analyzers.
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Materials and Methods
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QMS EVR assay materials for periods I, II and III evaluations were obtained from Thermo Scientific (Fremont, CA). Assay parameters for this user-defined test were programmed
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according to the manufacturer’s recommendation. 25
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Materials for LC-MS/MS assay
To support the method comparison evaluation, EVR and zinc sulfate monohydrate was
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purchased from Mass Supply Company (Highlands Ranch, CO). EVR-d4 was obtained from
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Toronto Research Chemicals (Toronto, Canada). HPLC grade methanol was purchased from
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VWR Scientific (Radnor, PA). Calibrator materials were obtained from ChromSystems (6PLUS1 Multilevel Whole Blood Calibrator Set, Gräfelfing, Germany) and quality control
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materials were purchased from UTAK Laboratories (Whole Blood Toxicology Control Level
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1&2, Valencia, CA).
Methods Validations and Comparisons QMS assay QMS EVR is a turbidimetric immunoassay. Sample preparation was initiated by mixing 300 L of sample (patient whole blood) with 350 L of methanol and 50 L of a precipitation reagent. After vortexing for 35 seconds and centrifugation for 8 minutes at 13,000 xg, 350 L of the
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ACCEPTED MANUSCRIPT supernatant was transferred to sample cups. Drug in the supernatant and drug coated on microparticle compete for the limited number of antibody binding sites. If EVR is absent in the sample supernatant, EVR-coated microparticles are agglutinated in the presence of antibody reagent. Agglutination is measured photometrically. If EVR is present, agglutination is partially
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inhibited depending on EVR concentration. Thus, the agglutination rate is inversely proportional
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to EVR concentration. Calibrators range was from 0, 1.5, 3.0, 6.0, 12.0 and 20 ng/mL. Precision
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and comparison studies were performed with de-identified patient samples. In establishing accuracy, a de-identified patient sample with a high EVR concentration was diluted with whole
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blood hemolysate and assayed five times. For Period 1, samples from renal transplant patient
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samples from were re-analyzed the following time periods post collections: Wake Forest, 1 to 2 weeks., ARUP, 2 to 3 months., and University of Colorado, 2 to 6 months., for Period II, ARUP
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samples within 2 to 3 months, and for Period III, from Wake Forest, within 1 to 2 weeks. These
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samples underwent 2 to 4 freeze and thaw cycles.
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Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay The EVR LC-MS/MS method was performed according to the recent publication by Johnson-
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Davis et al. 2015. 6 Data analysis was performed using Analyst® software and the concentration
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of EVR was calculated by applying the ratio of the peak areas of EVR to the corresponding internal standard. The peak area ratio from a patient sample was then compared to a calibration curve formed by a series of calibrators of known concentrations within the same matrix. Statistical analysis (Deming Regression Analysis and Bland-Altman Plot) was performed by the Biostatistics Department of Wake Forest School of Medicine. Results and Discussion
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ACCEPTED MANUSCRIPT The study may be readily divided into three sequential periods: Period I 2012-2013 - QMS assays comparison with DxC, AU680 and Indiko using deidentified samples from Wake Forest, ARUP and University of Colorado Period II 2013-2014 - comparison of LC-MS/MS with AU 680 and Indiko using de-
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identified samples from ARUP only
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Period III 2015-2016 - comparison of AU680 and AU5800 analyzers using de-identified
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samples from Wake Forest only.
Periods I and III studies of QMS assays by Indiko, DXc, AU680, and AU5822
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Linearity data of Periods I and III studies of Indiko, DxC, AU680, and AU5800, summarized in
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Table 1, showed analytical measuring ranges of 0.51 to 20 ng/mL with slopes ranging from 1.002 to 1.025. The lower limit of analytical measurement range was established as the limit of
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detection, determined by repeated dilution of the low calibrator, 1.5 ng/mL with imprecision
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<20%. Limit of quantitation for patient EVR monitoring was set at the low calibrator of 1.5 ng/mL. Calibration stabilities ranged from 1 day for DxC, different from the manufacturer’s
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claim, to 5 days for Indiko, AU680 and AU5800. Accuracy was established by dilution of a de-
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identified patient sample with high EVR concentration, and by comparison studies of Indiko, AU680 and LC-MS/MS. A de-idenified patient sample with EVR concentration of 13.6 ng/mL,
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diluted with whole blood hemolysate to low concentration of 1.8 ng/mL, and these were assayed five times. The corresponding concentrations, recoveries and CVs of these two samples sets analyzed by Indiko, DxC and AU 680 ranged from: 11.99 – 14.21 and 1.81 – 1.94 ng/mL., 98 101.56 and 93 - 115%., and 3.75 - 4.5 and 18.50 – 20.85 %. Day-to-day precision studies, shown in Table IB, showed CVs of 2.6 to 8.9% for the low, medium and high controls. Period I comparison studies of the three analyzers, shown in Table 2, included 107 de-identified
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ACCEPTED MANUSCRIPT transplant samples with concentrations ranging from <1.5 to 13.6 ng/mL. Deming regression showed the 5 slopes ranging from 1.000 to 1.076, acceptable intercepts and correlations, Sy/x ranging from 0.49 to 0.78. Period III, performed in early 2015 after the second automation update, compared AU 680 to AU5822 analyzers, showing slope of 1.007. This later comparison
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Period II study of LC-MS/MS and QMS assays comparison
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was performed with a limited number of samples.
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Period II study included 104 de-identified EVR patient samples obtained from ARUP. The samples were analyzed by LC-MS/MS, and later re-analyzed by QMS EVR assays using Indiko
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and AU680 in our lab. Performance data and statistical analyses using Deming regression and
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Bland-Altman plots are shown in Figures 1a-f and Table 3. Comparison studies between LCMS/MS vs AU680, LCMSMS vs Indiko, and AU 680 vs Indiko showed slopes of 1.046, 1.086
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and 1.035 respectively., and the corresponding mean differences, as shown by the Bland-Altman
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plots, were: 0.05 (95% CI -1.83 to 1.84), -0.45 (95% CI -2.74 to 1.83) and -0.47 (95% CI -1.63 to 0.85). Mean concentrations for LC-MS/MS, AU680 and Indiko were 5.403, 5.393, 5.849
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ng/mL respectively and Sy/x ranged from 0.53 to 0.74 ng/mL. Indiko results showed a positive
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bias, with the mean concentration of 5.849. The differences of immunoassay platforms and LCMS/MS, the time elapsed between the two assays, and sample handling might have contributed
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to the data scattering, as shown by Deming regression and Bland-Altman plots. Within the period II evaluation, 42 de-identified samples from 11 renal transplant patients were monitored over various time periods, as shown by Supplemental Table 3. EVR concentrations of renal transplant patients were 88% within the recommended range of 3-4 to 6-8 ng/mL as suggested by Hesselink.13
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ACCEPTED MANUSCRIPT QMS EVR monitoring proficiency performance, as shown by Supplemental Table 4, was evaluated by our enrollment with the College of American Pathologists (CAP) Survey, using AU680 for 2013-2014, and AU5800 for 2015 - 2016. Standard Deviation Index (SDIs) and the means ranged from:-1.7, Wake Forest (WF) and QMS means of 11.1 and 11.98., to +1.8, WF
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and QMS means of 6.0 and 5.05 respectively. These differences would most likely not have
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changed EVR TDM.
For 2014-2016 CAP EVR annual surveys, there were two series (EV-A and EV-B) with three
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concentrations – low, medium and high. For the low concentration samples, the reported ranges (ng/mL) of LC-MS/MS assays, PETINTA assays, and difference ranges of the two assays were:
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2.0 to 2.2, non-detected (ND) to 2.0, and ND to 2.0 respectively., for medium concentrations,
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7.9 to 8.5, 5.1 to 5.5, and 2.4 to 3.1., and for high concentrations, 16.8 to 21.6, 12.4 to 14.4 and 4.3 to 7.1 respectively. In assessing EVR TDM, the difference range of medium concentrations
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of 2.4 to 3.1 would be of interest, and might be explained by the formulation of the assay
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calibrators at about 70% of the gravimetrically-spiked samples as explained in a later section.
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Comparison of this study with previous publications In two previous publications comparing immunoassays as shown by Supplemental Table 1,
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Buthiau et al. compared FPIA/Innofluor to QMS Indiko, showing a slope of 0.81, possibly due to different immunoassay chemistry. 19 However, Mori et al. compared FPIA/Innofluor to Nanopia/QMS, showing an acceptable slope of 0.966. 22 For comparisons of LC-MS/MS assays to QMS immunoassays, findings from previous five publications, as shown by Supplemental Table 2, showed slopes ranging from 0.8 to 1.277. Hopfer compared QMS EVR performed on Architect (Abbott) analyzers to LC-MS/MS for monitoring 250 samples from 169 renal, heart, lung, and liver transplant patients. 20 Mean 10
ACCEPTED MANUSCRIPT concentrations for LC-MS/MS and QMS were 4.8 and 6.3 ng/mL respectively. Above therapeutic concentrations by QMS accounted for 69% of samples which were shown to be therapeutic by LC-MS/MS. This lack of agreement resulted in the authors’ suggestion that these assays did not yield interchangeable EVR concentrations. Shu et al compared LC-MS/MS to
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EVR QMS assay by Vitros using 34 samples.21 This later study showed a “high” slope of 1.271,
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but the authors concluded that the assay was “reliable and reproducible”.18 The results of these
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two studies are different from findings of two other studies by Dasgupta et al.3 and Buthaiau.19 Dasgupta ‘s compared LC-MS/MS to QMS assay by using Hitachi 917, showing a slope of 1.11. Buthaiau compared the QMS EVR assay using Indiko to UPLC-MS/MS and an
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3
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Innofluor/FPIA assay (Seradyn Inc) for 120 samples from 74 transplant patients (35 kidney, 30 liver and 9 heart).19 The study showed QMS EVR on the Indiko was reliable with acceptable
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accuracy when compared to UPLC-MS/MS, UPLC-MS/MS to QMS by Indiko, showing a slope
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of 0.99.while the Innofluor assay (FPIA, Abbott, recently discontinued) over-recovered by about 21%,19 similar to a 24% higher measurement observed in an earlier published study. 22
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These results are similar to our Period II comparison study of using Indiko and AU680 analyzers,
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and LC-MS/MS showed more comparable and acceptable slopes ranging from 1.046 to 1.086. The difference in slopes might be attributable to multiple factors – sample types, and calibration
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and variability of the study methods including LC-MS/MS. This was one of the findings from the long-term cross-validation Zortracker Study,26 as well as one of the conclusions of Holt’s presentation at the 2014 EVR Consensus Conference. 14 Further, the differences between these studies might be partially attributed to QMS EVR assay calibration. As described by the manufacturer in the QMS EVR package insert, “calibrators and controls are prepared gravimetrically by adding EVR to human whole blood. According to the
11
ACCEPTED MANUSCRIPT manufacturer, to minimize bias between QMS and LC-MS/MS results, value assignment of calibrators and controls were based on a set of clinical trough EVR patient samples and would result in “under-recovery “at 70% of the gravimetrically-spiked samples.13 This might explain the CAP EVR surveys summarized in Supplemental Table 4 showing negative basis for the
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QMS assay group with testing EVR-spiked samples rather than patient-derived samples.
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According to the manufacturer, EVR metabolites cross-reactivities ranged from 2 – 63%.25
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However, a recent study showed the addition of two specific EVR metabolites did not affect the Zortracker survey results. 26 In assessing the EVR TDM performance from multi-centers,
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Schniedewind et al. sent EVR samples from a prospective clinical trial to participating
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laboratories: LC-MS/MS (n=6 to13) and QMS (n = 4-8 ) assays.26 In order to test for interference, two metabolites, 39-O-desmethyl and 46-hydroxy EVR were added to two
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individual pools. The analyses of these two sample pool samples (Feb. 2013 to Feb. 2014),
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showed comparable means (ng/mL) and ranges for 107 LC-MS/MS and 77 QMS assays samples as follows: 5.31 and 2.9 – 7.8 ng/mL, and 5.20 and 4.0 – 6.8 ng/mL respectively. Thus, the
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authors suggested that the two added metabolites did not interfere with the assays.
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Shortly after this publication, the influence of laboratory practices on immunosuppressant
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monitoring variability was investigated using data generated by an immunosuppressants survey sponsored by Novartis and under the auspice of IATDMCT.27 From the 2013 survey of 76 participating laboratories in 14 countries, the variability was due to differences in lab procedures, lack of standardization in pre-analytics, use of inappropriate reference materials, and noncompliance to accepted international guidelines. This might be improved by standardized operational procedures and personnel proficiency. Limitations 12
ACCEPTED MANUSCRIPT For the period I and II studies, de-identified samples and their LC-MS/MS concentrations were mostly provided by outside laboratories, and our QMS measurements were “delayed” for several weeks relative to the LC-MS/MS measurements. This delay, with time period comparable to that of performing CAP survey, did not contribute to significant variability. As shown by CAP EVR
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surveys, LC-MS/MS and EVR QMS assays of “spiked “ survey samples showed the “expected “
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significant differences.
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Conclusions: EVR may be monitored by the QMS assay using four analyzers - Indiko, DxC, AU680 and AU5822 with adequate limit of detection and acceptable precision, showing high
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correlation of the four analyzers. Over the past 3.5 years, we have found these four analyzers
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offered TDM EVR monitoring suitable for renal and liver transplant recipients.
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ACCEPTED MANUSCRIPT Acknowledgements: This study was supported by an instrument/reagent evaluation grant from Thermo Fisher Scientific. The co-authors greatly appreciated the provisions of left-over EVR samples and
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guidance by colleagues from University of Colorado.
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ACCEPTED MANUSCRIPT References: 1. Kirchner GI, Meier-Wiedenbach I, Manns MP. Clinical pharmacokinetics of everolimus. Clin Pharmacokinetic., 2004;43:83-95. 2. Kuhn B, Jacobsen W, Christians U, Benet LZ, Kollman PA. Metabolism of Sirolimus and
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its Derivative Everolimus by Cytochrome P450 3A4: Insights from Docking, Molecular
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Dynamics, and Quantum Chemical Calculations. J Med Chem;2001:44:2027-34.
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3. Dasgupta A, Davis B, Chow L. Evaluation of QMS Everolimus Assay using Hitachi 917 analyzer: Comparison with Liquid Chromatography/Mass Spectrometry. Ther Drug
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Monit. 2011;33:149-54.
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4. Billaud EM. Clinical pharmacology of Everolimus with focus on implication on Therapeutic Drug Monitoring. Accessed: January 10, 2016.
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https://www.iatdmct.org/resources/presentations/426-rsc-pres-isd-20140911.html.
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5. Gabardi S, Baroletti SA. Everolimus: a proliferation signal inhibitor with clinical
2010;30:1044-56.
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applications in organ transplantation, oncology, and cardiology. Pharmacotherapy.
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6. Johnson-Davis KL. Juenke JM, Thomas RL, Bradshaw T. Everolimus method comparison between Waters MassTrakTM immunosuppressants XE(IUO) kit and an in-
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house laboratory developed LC-MS/MS method in renal transplant patients. Ann Clin Lab Sci. 2015;45:27-31. 7. Picard N, Bergan S, Marquet P, van Gelder T, Wallemacq P, Hesselink DA, Haufroid V. Pharmacogenetic biomarkers predictive of the pharmacokinetics and pharmacodynamics of immunosuppressive drugs. Ther Drug Monit. 2016;38(S1):S57-S69.
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ACCEPTED MANUSCRIPT 8. Strom T, Haschke M, Zhang YL, et al. Identification of Everolimus metabolite Patterns in Trough Blood Samples of Kidney Transplant Patients. Ther Drug Monit. 2007;29:5929. 9. ITADMCT – Immunosuppressive drugs consensus conference presentations. September,
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2014, Stuttgart, Germany. Accessed: January 10, 2016.
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https://iatdmct.org/resources/presentations/426-rsc-pres-isd-20140911.html
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10. Shipkova M, Hessleink DA, Holt DW, Billaud EM, van Gelder T, Kunickt PK, et al. Therapeutic Drug Monitoring of Everolimus: A Consensus Report. Ther Drug Monit.
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2016;38:143-169.
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11. Seger C, Shipkova M, Christians U, Billaud E, Wang P, Holt D, Brunet M, Kunicki PK, Pawiński T, Langman LJ, Marquet P, Oellerich M, Wieland E, Wallemacq P.
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Assuring the Proper Analytical Performance of Measurement Procedures for
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Immunosuppressive Drug Concentrations in Clinical Practice: Recommendations of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology
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Immunosuppressive Drug Scientific Committee. Ther Drug Monit. 2016;38:170-189.
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12. Felipe C, Oliveira N, Hannun P, de Paula MI, Tedisco-Silva H, Medina-Pestana JO. Pharmacokinetics and long-term safety and tolerability of Everolimus in renal transplant
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recipients converted from cyclosporine. Ther Drug Monit. 2016;38:64-72. 13. Hesselink DA. Practice of TDM of Everolimus: An overview: Accessed: January 10, 2016. https://www.iatdmct.org/resources/presentations/426-rsc-pres-isd-20140911.html. 14. Holt DW. Analytical aspects of the determination of Everolimus concentrations. Accessed: January 10, 2016. https://www.iatdmct.org/resources/presentations/426-rscpres-isd-20140911.html.
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ACCEPTED MANUSCRIPT 15. Wong SHY, Johnson-Davis K, Garrison K Evaluation of Everolimus QMS assay by using Thermo Scientific Indiko, Beckman DxC, and AU680 analyzers. Clin Chem. 2013;59:A135. 16. Wong C, Cheng D, Naqvi H, Yokobata. QMS EVR assay for Beckman Coulter AU480,
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AU680, AU5800 Clinical Chemistry Analyzers. Clin Chem. 2014;60:S150.Baldelli S,
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Crippa A, Gabrieli R, et al. Comparison of the Innofluor Certican assay with HPLC-UV
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for the determination of Everolimus concentrations in heart transplantation. Clin Biochem. 2006;39:1152-1159
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17. Baldelli S, Crippa A, Gabrieli R, et al. Comparison of the Innofluor Certican assay with
Clin Biochem. 2006;39:1152-1159
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HPLC-UV for the determination of Everolimus concentrations in heart transplantation.
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18. Wong C, Cheng D, Thao A, Ye L. QMS Tacrolimus assay for Beckman Coulter AU480,
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AU680, AU5800 Clinical Chemistry Analyzers. Clin Chem. 2014;60:S145. 19. Buthiau D, Bargnoux A, Badiou S, Sutra T, Dupuy A, Pageaux G, Mourad G, Cristol J.
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Evaluation of QMS Everolimus assay using Indiko analyzer: comparison with an ultra-
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performance liquid chromatography-tandem mass spectrometry method. Ther Drug Monit. 2015;37:275-278.
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20. Hoffer E, Kurnik D, Efrati E, Scherb I, Karasik M, Ring G, Bentur Y. Comparison of Everolimus QMS immunoassay on Architect ci4100 and liquid chromatography/mass spectrometry: Lack of agreement in organ-transplanted patients. Ther Drug Monit. 2015;37:214-219.
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ACCEPTED MANUSCRIPT 21. Shu I, Wright AM, Chandler WL, Bernard DW, Wang P. Analytical performance of QMS Everolimus assay on Ortho Vitros 5,1 FS Fusion analyzer: Measuring EVR trough levels for solid organ transplant recipients. Ther Drug Monit. 2014;36:264-268. 22. Mori K, Kimura S, Matsui M, Suehisa E, Hidaka Y, Fufushima N. Latex-enhanced
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turbidimetric immunoassay for Everolimus in whole blood using the Nanopia TDM EVR
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assay with the JCA-BM6010 automatic analyzer. Ther Drug Monit. 2014;36:677-680.
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23. McMillin GA, Johnson-Davis K, Dasgupta A. Analytical performance of a new liquid chromatography/tandem mass spectrometric method for determination of Everolimus
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concentrations in whole blood. Ther Drug Monit. 2012;34:222-226
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24. QM Zimmerman MK, Friesen LR, Nice A, Vollmer PA, Dockery EA, Rankin JD, Zmuda K, Wong SHY. Multi-center evaluation of analytical performance of the Beckman Coulter
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AU5822 chemistry analyzer. Clin Biochem.2015;48:881-885.
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25. Everolimus (Package insert). Fremont, CA Thermo Fisher, 2010. 26. Schniedewind B, Niederlechner S, Galinkin JL, Johnson-Davis KL, Christians U, Meyer
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EJ. Long-term cross-validation of Everolimus therapeutic drug monitoring assays: The
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Zortracker study. Ther Drug Monit. 2015;37:296-303. 27. Christians U, Vinks AA, Langman LKJ, Clarke W, Wallemacq P, van Gelder T, Renjen
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V, Marquet P, Meyer EJ. Impact of Laboratory practices on interlaboratory variability in Therapeutic Drug Monitoring of immunosuppressive drugs. Ther Drug Monit. 2015;37:718-724.
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ACCEPTED MANUSCRIPT Table 1 - Period I and III QMS EVR assay performance Linearity of QMS EVR assay by Indiko, AU680, DxC and AU5800 Analytical
Slope
R2
Intercept
measuring
ng/mL
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ranges, ng/mL
Indiko
0.53-20
1.014
0.121
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Period I
AU680
0.51 – 19
1.025
0.105
0.998
0.51-20
1.023
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DxC
0.195
0.76 – 20.0
0.674
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1.002
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Period III AU5822
0.997
19
0.998
0.968
ACCEPTED MANUSCRIPT Table 1B: Period I and III Day-to-Day Precision studies for Indiko, DxC, AU680 and AU5800(n
= 20 and study duration = 2 weeks). Sample
Mean conc.(ng/mL)
CV, %
Analyzer Period I
DxC
Medium
7.81
High
15.25
3.1
Low
4.72
3.6
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High
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Medium
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Low
5.4
7.70
4.5
15.56
2.6
4.20
3.1
7.80
3.4
14.90
3.5
Low
4.10
8.7
Medium
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8.20
8.9
High
14.80
4.4
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AU5822
6.8
Period III
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Medium
AU680
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4.00
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Indiko
Low
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Intercepts, ng/mL
DxC vs. Indiko
107
Period I 1.073 (1.019 to 1.127)
AU680 vs. Indiko
107
1.076 (1.022 to 1.134)
DxC vs. AU680
107
1.000 (0.961 to 1.038)
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Period III 1.007 (0.957 to 1.057)
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0.434 (0.192 to 0.843)
0.945
0.71
0.462 (0.221 to 0.814)
0.962
0.78
-0.053 (-0.262 to 0.154)
0.981
0.49
-0.265 (-0.622 to 0.038)
0.980
0.53
-0.802 (-1.012 to -0.593)
0.952
0.61
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AU680 vs AU5822
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104
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AU680 vs Indiko
Sy/x ng/mL
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Slopes
Period II 1.035 (0.983 to 1.087)
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Analyzers,
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Table 2 – Correlation data of Immunoassays analyzers comparison with de-identified transplant samples: for Period I, from ARUP, University of Colorado and Wake Forest., for Period II from ARUP., and for Period III from Wake Forest.
ACCEPTED MANUSCRIPT Table 3: Summaries of comparison of LC-MS/MS with QMS EVR assays of de-identified samples from ARUP for the Period II study
Intercepts ng/mL
R
n
Mean LCMS/MS ng/mL
Mean QMS AU680 ng/mL
Mean QMS Indiko ng/mL
Sy/x ng/mL
LC-MS/MS vs AU680
1.046 (0.981 to 1.111)
-0.256 (-0.561 to 0.042)
0.945
104
5.403
5.393
--
0.74
LC-MS/MS vs Indiko
1.086 (1.025 to 1.147)
-0.019 (-0.332 to 0.293)
0.924
104
5.403
--
5.849
0.71
AU680 vs Indiko
1.035 (0.983 to 1.087)
-0.265 (-0.622 to 0.038)
0.980
104
5.393
5.849
0.53
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Slopes
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Slope
Intercept, ng/mL
DxC vs. Indiko AU680 vs. Indiko DxC vs. AU680
107 107 107
This study Period I 1.073 1.076 1.000
0.434 0.462 -0.053
0.945 0.962 0.981
DxC vs AU680
104
Period II 1.035
-0.265
0.980
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Period III 1.007
-0.802
0.952
Other publications 0.81
-0.09
0.94
0.946
NA
0942
120
91
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Innofluor/FPIA vs QMS Indiko, Buthaiau (19) Innofluor/FPIA vs Nanopia/QMS Mori (22)
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AU680 vs AU5822
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Analyzers, author (reference)
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n
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Supplemental Table 1 – Correlation data of Immunoassays analyzers comparison of Periods I, II and III of this study with de-identified transplant samples (refer to table 2 for sample sources) and other recent publications
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NA, Not available
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n
Mean LCMSMS
Mean QMS
Comments
Period II of this study 1.046
0.945
104
5.403
5.393
Refer to the results and discussion of this paper
1.086
0.924
104
5.403
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5.849
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LC-MS/MS vs AU680
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90
NA
NA
0.95
120
5.3
5.2
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Wong et al This study
Slopes
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Method comparison
0.8
0.66
250
4.8
6.3
LC-MS/MS vs Nanopia/QMS
0.960
0.946
91
6.41
6.47
LC-MS/MS vs QMS(Vitros)
1.271
0.880
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NA
NA
LC-MS/MS vs QMS (Vitros)
1.277
0.912
8 (liver only)
NA
NA
LC-MS/MS vs Indiko
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References
Other publications
Buthaiau et al, 2015 (19)
UPLC-MS/MS vs QMS Indiko
0.99
Hopfer et al. 2015 (20)
LC-MS/MS vs QMS Architect
Mori et al 2014 (22) Shu et al 2014 (21)
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0.92
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1.11
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LC-MS/MS vs QMS Hitachi
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Dasgupta et al (3)
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Adequate sensitivities, specificity and analytical measurement range QMS - Reliable and reproducible performance QMS - Significant positive bias – not used interchangeably Nanopia/QMS Suitable for routine use QMSSatisfactory analytical performance QMS Satisfactory analytical performance
ACCEPTED MANUSCRIPT Supplemental Table 3 - Period III EVR concentrations by using AU 5822 for 42 de-identified samples from 11 renal transplant patients
28/6 21/6
69/5 34/5
51/5
13/6 4/3
20/6 6/2*
26/6 7/<2*
34/4 8/<2*
27/4 16/5 35/5 49/4
55/4 28/5
** Above therapeutic
51/5 28/7
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ACCEPTED MANUSCRIPT Supplemental Table 4 – CAP EVR survey results from Wake Forest (WF) and peer groups from 2013 to 2016 CAP surveys for QMS EVR PETINA (PET)** and LC-MS/MS (LCMS). EVR concentration, ng/mL
LC-MS
EV-A
1 2 3
12.0 <2.0 5.0
12 1.5 5.2
17.6 2.1 8.4
<2.0 5.0 12.0
SDI *** -1.2 -0.4
EV-B
4 5 6
<2.0 5.0 12.0
1.1 5.2 11.3
2.1 8.2 17.7
4.9 11.1 <2.0
-1.5 -1.7 ***
1 2 3
5.0 13.0 3.0
EV-B
4 5 6
15.0 2.0 6.0
1.1 1.2 1.8
5.52 11.98 ***
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EV-A
SDI -0.1 -1.2 1.4
PET Mean 5.1 13.7 1.6
SD 0.73 0.58 1.02
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2015 WF
LC-MS Mean 8.18 20.16 2.14
SD 1.21 2.93 0.44
13.88 1.14 5.05
1.02 0.10 0.54
20.98 2.18 8.18
3.06 0.34 1.11
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Sample No.
PET Mean *** 5.5 12.44
SD *** 0.46 0.99
LC-MS Mean 2.0 7.9 16.76
SD 1.29 1.12 2.34
0.41 0.53 ***
8.5 17.77 2.17
1.04 2.05 0.34
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PET
2014 WF
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2013** WF
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CAP
Sample No.
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CAP
2016 WF 16.0 <2.0 6.0
SDI 1.3 1.0
PET Mean 14.42 1.62 5.49
SD 1.24 0.71 0.53
LC-MS Mean 20.57 2.22 8.51
SD 2.34 0.36 1.23
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* Standard Deviations (SDs) of 2013 surveys were no longer available as a result of the normal practice of keeping survey results for only two recent years
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**QMS results were grouped under PETINA(PET) by CAP *** No mean and SD reported for results of less than 10 reporting labs SDI – Standard deviation Index = (WF EVR concentration – EVR mean concentration)/EVR SD
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ACCEPTED MANUSCRIPT Figure Legends Figure 1 Deming Regression and the Bland-Altman bias plots for comparisons of QMS EVR assays of 104 EVR patient samples from ARUP. The inner blue line of the Bland-Altman plots represents the mean difference of the two assays, and outer blue lines are the 95% CI of the mean difference.
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Figures 1a and 1b show the Deming Regression and the Bland-Altman bias plot for comparison of EVR assays by LC-MS/MS and QMS Indiko.
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Figures 1c and 1d show the Deming Regression and the Bland-Altman bias plot for comparison of EVR assays by LC-MS/MS and QMS AU680.
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Figures 1e and 1f show the Deming Regression and the Bland-Altman bias plot for comparison of QMS EVR assays using AU680 and Indiko.
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Figure 1d
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Figure 1e
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Figure 1f
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