Irina Baburina

An automated nanoparticle-based homogeneous immunoassay for determining docetaxel concentrations in plasma.

Background: Docetaxel (Taxotere) (DTX) is a widely used chemotherapy agent used in many regimens for the treatment of solid tumors, for example breast cancer, non–small cell lung cancer, gastric, prostate, and head and neck cancers. This drug meets the criteria for therapeutic dose management, in that it is associated with high pharmacokinetic variability and dose-limiting toxicity; it has a narrow therapeutic window, and there is a significant pharmacokinetic–pharmacodynamic relationship. Measures of exposure and area under the time–concentration curve have been associated with both toxicity and outcomes, making therapeutic dose management for this drug an unmet clinical need. The current methodologies for measuring DTX are based on physical methods, making the analysis less available and costly. An automated immunoassay has been developed to provide greater access to DTX dose management. Methods: A DTX immunoassay (MyDocetaxel) has been developed using a generic nanoparticle turbidimetric method that can be used on a wide variety of automated clinical chemistry analyzers including the Beckman Coulter AU400 and AU640 instruments, which were used in this study. The assay is based on a competitive assay format using a selective DTX monoclonal antibody. Clinical Laboratory Standards Institute protocols for establishing manufacturers claims were used to verify performance. Testing at 3 clinical laboratories was undertaken using the same protocols for laboratory validation of precision, accuracy, and linearity. Method comparison (n = 89) was done using samples collected from patients on DTX therapy. The comparative method was LC–MS/MS validated according to Food and Drug Administration guidance on bioanalytical methods. Institutional review board approval was obtained for prospective collection of samples from patients on DTX therapy. Results: The assay on the AU400 uses 2 &mgr;L of sample, provides the first result in 9.0 minutes and can generate 400 determinations per hour. Internal studies established a lower limit of detection ⩽25 ng/mL and a lower limit of quantitation ⩽30 ng/mL. Additional studies demonstrated no interference from coadministered drugs, major metabolites, or related compounds. Linearity from 50 to 1000 ng/mL was validated. Method comparisons between laboratories and to the physical method gave slopes: 1 ± 0.5, intercepts: < 2.0 ng/mL, R > 0.99, with the range of DTX concentrations measured by the assay 31–9754 ng/mL, with a mean of 689 ng/mL. In all 3 laboratories, the coefficient of variation percentage for repeatability ranged from 0.8% to 6.2% and the within-laboratory precision ranged from 1.4% to 10.1%. Conclusions: This immunoassay is suitable for quantifying DTX in plasma with advantages of small sample size, no sample pretreatment, and the ability to be applied to a wide range of clinical analyzers. With the validation of this method, the application of DTX testing in clinical practice may gain wider acceptance for individualizing patient DTX dosing.

Development and Evaluation of a Nanoparticle-Based Immunoassay for Determining Paclitaxel Concentrations on Routine Clinical Analyzers

Background: Paclitaxel (PTX; Taxol, Abraxane) is used in many regimens for breast cancer, non–small cell lung cancer (NSCLC), and ovarian cancer. Multiple studies have demonstrated that PTX exhibits a greater than 10-fold interpatient variability of clearance rates when patients are dosed according to body surface area (BSA). Pharmacokinetic and pharmacodynamic relationships have been elucidated from BSA-based dosing. PTX is a candidate for dose management, and studies have shown that therapeutic dose management (TDM) is feasible and may provide improved outcomes for patients undergoing treatment. Methods: A PTX immunoassay (MyPaclitaxel) has been developed, which employs a novel PTX monoclonal antibody in a nanoparticle-based turbidimetric assay in a competitive format. Precision, accuracy, and linearity were evaluated by Clinical Laboratory Standards Institute protocols at 3 laboratories on the Olympus AU400 analyzer. Method comparison was done versus a validated high-performance liquid chromatography–tandem mass spectroscopy method using samples (n = 119) collected from patients on PTX therapy. Results: The assay requires 8 &mgr;L of plasma sample and can produce 400 determinations per hour. The response curve is based on a 6-point nonlinear curve fit and has a range of 0-320 ng/mL, extended to 3200 ng/mL with 10-fold autodilution. Three controls and 4 patient pools were used in precision studies. For all samples across 3 sites, repeatability coefficient of variation percentages ranged 0.9%-4.9%, and within-laboratory coefficient of variation percentages were 1.0%-4.2% with standard curve stability up to 24 days. Linearity was demonstrated over the linear range. Lower limits of detection and quantitation were 11 and 19 ng/mL, respectively. Method comparison results were analyzed by Deming regression, demonstrating a slope = 1.002 and intercept = −3.029 and an R = 0.996. The PTX samples ranged from 24 to 3164 ng/mL with a mean of 745 ng/mL. Conclusions: The analytical performance of an automated immunoassay for PTX has been validated and may serve as a useful tool for TDM of this drug.

An Automated Homogeneous Immunoassay for Quantitating Imatinib Concentrations in Plasma.

Background: Imatinib pharmacokinetic variability and the relationship of trough concentrations with clinical outcomes have been extensively reported. Although physical methods to quantitate imatinib exist, they are not widely available for routine use. An automated homogenous immunoassay for imatinib has been developed, facilitating routine imatinib testing. Methods: Imatinib-selective monoclonal antibodies, without substantial cross-reactivity to the N-desmethyl metabolite or N-desmethyl conjugates, were produced. The antibodies were conjugated to 200 nm particles to develop immunoassay reagents on the Beckman Coulter AU480 analyzer. These reagents were analytically validated using Clinical Laboratory Standards Institute protocols. Method comparison to liquid chromatography tandem mass spectrometry (LC-MS/MS) was conducted using 77 plasma samples collected from subjects receiving imatinib. Results: The assay requires 4 µL of sample without pretreatment. The nonlinear calibration curve ranges from 0 to 3000 ng/mL. With automated sample dilution, concentrations of up to 9000 ng/mL can be quantitated. The AU480 produces the first result in 10 minutes and up to 400 tests per hour. Repeatability ranged from 2.0% to 6.0% coefficient of variation, and within-laboratory reproducibility ranged from 2.9% to 7.4% coefficient of variation. Standard curve stability was 2 weeks and on-board reagent stability was 6 weeks. For clinical samples with imatinib concentrations from 438 to 2691 ng/mL, method comparison with LC-MS/MS gave a slope of 0.995 with a y-intercept of 24.3 and a correlation coefficient of 0.978. Conclusions: The immunoassay is suitable for quantitating imatinib in human plasma, demonstrating good correlation with a physical method. Testing for optimal imatinib exposure can now be performed on routine clinical analyzers.

BIOMARKER STABILITY IN CSF: PRE-ANALYTICAL FACTORS IN A PROSPECTIVE COLLECTION

of the optical densities in ten repetitions of the standard curves was 5% for all standards. In the clinical part, at the cut off value 691 pg/mL, Ab 1-42 showed the sensitivity and the specificity of 69.3 %, and 88.9%, respectively, whereas at the cut off value 0.06, Ab 42/40 ratio showed even better sensitivity and specificity of 93.3%, and 100%, respectively. The area under the ROC curve for Ab 42/40 (0.974) was highly significantly larger compared to the AUC of the Ab 1-42 concentration ROC curve (0.827, p<0.0001). Conclusions: (a) the novel Ab1-40 and Ab1-42 ELISA assays characterize with very good analytical performance; (b) we reconfirm that the CSF Ab42/40 concentration ratio shows significantly better diagnostic performance compared to the CSF Ab1-42 concentration alone.

Reproducibility of a fully automated, chemiluminescent, beta-amyloid 42 assay

upon a detailed unified SOP for the INNO-BIA AlzBio3 (RUO; Innogenetics nv, Belgium) multiplexing xMAP assay. Using the same assay batch, a set of ten CSF pool samples and two buffer based control samples were analyzed using ready-to-use calibrators. A fresh aliquot of each sample was tested in quadruplicate in three independent assay runs (only 2 runs for center C). The goal of the study was to determine the with-in and interlaboratory variability of the assay for hTau, Ab 1-42, and P-Tau 181P after implementation of the unified SOP. Results: The CSF analyte concentrations showed a strong correlation between all centers (R> 0.95; regression center versus overall center mean). For the CSF samples, the total inter-center variability over the 8 runs performed (%CV) ranged between 12.4 22.9% (hTau), 8.1 13.1% (Ab 1-42), and 6.8 13.2% (P-Tau 181P). Across the 10 CSF samples, the mean within-laboratory variation ranged between 1.8% and 11% for all analytes. The variability observed for the control samples was even lower (max. 4.5%).Conclusions: Implementation of a unified SOP in the three laboratories resulted in an acceptable inter-laboratory variation of analyte concentrations for CSF and control samples. Careful documentation of the critical parameters of the test procedure and rigorous adherence to a detailed instruction of use clearly leads to highly comparable CSF analyte concentrations between experienced centers.

Performance of preliminary cut-points for progression from mild cognitive impairment to dementia of the Alzheimer's disease type using fully automated chemiluminescent beta-amyloid 42 and tau assays

P2-072 PERFORMANCE OF PRELIMINARY CUT-POINTS FOR PROGRESSION FROM MILD COGNITIVE IMPAIRMENT TO DEMENTIA OF THE ALZHEIMER’S DISEASE TYPE USING FULLY AUTOMATED CHEMILUMINESCENT BETAAMYLOID 42 AND TAU ASSAYS Adam Simon, Salvatore Salamone, William Clarke, George Green, Holly Soares, Leah Burns, Irina Baburina, Daniel Kozo, Jodi Courtney, Paul Contestable, Shari Jackson, Patience Ajongwen, AJ Simon Enterprises LLC., Yardley, Pennsylvania, United States; Saladax Biomedical, Inc., Bethlehem, New Jersey, United States; Johns Hopkins University School of Medicine, Baltimore, Maryland, United States; Bristol Myers Squibb, Princeton, New Jersey, United States; Bristol Myers Squibb, Wallingford, New Jersey, United States; Bristol-Myers Squibb, Wallingford, New Jersey, United States; Saladax Biomedical, Bethlehem, Pennsylvania, United States; Ortho Clinical Diagnostics, Rochester, New York, United States; Ortho Clinical Diagnostics, Rochester, New York, United States. Contact e-mail: ssalamone@saladax.com

Initial evaluation of fully automated, chemiluminescent beta-amyloid 42 and tau assays

P1-179 INITIAL EVALUATION OF FULLYAUTOMATED, CHEMILUMINESCENT BETA-AMYLOID 42 AND TAU ASSAYS Adam Simon, Irina Baburina, William Clarke, George Green, Holly Soares, Salvatore Salamone, Daniel Kozo, Jodi Courtney, Paul Contestable, Shari Jackson, Patience Ajongwen, A.J. Simon Enterprises Llc., Yardley, Pennsylvania, United States; Saladax Biomedical, Bethlehem, Pennsylvania, United States; Johns Hopkins University School of Medicine, Baltimore, Maryland, United States; Bristol Myers Squibb, Princeton, New Jersey, United States; Bristol Myers Squibb, Wallingford, New Jersey, United States; Saladax Biomedical, Inc., Bethlehem, Pennsylvania, United States; Ortho Clinical Diagnostics, Rochester, New York, United States; Ortho Clinical Diagnostics, Rochester, New York, United States. Contact e-mail: ibaburina@saladax.com

Stability performance of a fully automated, chemiluminescent, beta-amyloid 42 assay

P1-137 STABILITY PERFORMANCE OFA FULLY AUTOMATED, CHEMILUMINESCENT, BETA-AMYLOID 42 ASSAY Karen Ackles, George Green, Holly Soares, Flora Berisha, Robert Neely, Irina Baburina, Salvatore Salamone, Daniel Kozo, Shari Jackson, Anne Tweedie, Deb Byrne, Lisa DiMagno, Ortho Clinical Diagnostics, Rochester, New York, United States; Bristol Myers Squibb, Princeton, New Jersey, United States; Bristol Myers Squibb, Wallingford, New Jersey, United States; Bristol Myers Squibb, Lawrenceville, New Jersey, United States; Bristol-Myers Squibb, Princeton, New Jersey, United States; Saladax, Bethlehem, Pennsylvania, United States; Saladax Biomedical, Inc., Bethlehem, Pennsylvania, United States; Saladax Biomedical, Bethlehem, Pennsylvania, United States. Contact e-mail: kackles@its.jnj.com