Abstract Background Therapeutic drug monitoring (TDM) of antipsychotic blood levels is strongly recommended (clozapine and olanzapine) and recommended (risperidone, aripiprazole, and quetiapine) by ASCP/AGNP Consensus Statement. For effective TDM, it is paramount to understand both the stability of the antipsychotic drugs in serum and plasma, as well as any differences in levels due to sample matrix to ensure robust concentration determinations. While clozapine levels are considered equivalent in serum and plasma, we included in our study four additional antipsychotics. Methods Whole blood samples from ten patients taking clozapine, risperidone, aripiprazole, olanzapine, or quetiapine were collected in one red top tube and in one purple top tube according to an IRB-approved protocol. Samples were then processed within 2 hours of collection to obtain matched serum and K2EDTA plasma samples from each of the patients. CE marked immunoassays for the measurement of clozapine (also FDA cleared), total risperidone/paliperidone, total aripiprazole, olanzapine, and quetiapine were used to quantitate drug concentrations. Stability of the samples was assessed at 4°C, ambient room temperature (ART), and frozen at -80°C, with respect to their Day 0 concentration by measurement of six replicates at each timepoint using the immunoassays. Results Comparison of Day 0 values between serum and plasma for the same patient showed ±9% bias between sample matrices for all drugs, except olanzapine which showed differences up to ±15%. Average bias between serum and plasma was ≤-5% (olanzapine: -9%). All drugs were stable (±15% deviation from Day 0; olanzapine up to ±23%) in serum and in plasma for 7 days at both 4°C and ART and for up to 8 months frozen. Conclusions This study demonstrated that (1) the antipsychotic drugs clozapine, risperidone, aripiprazole, olanzapine, and quetiapine have sufficient stability in both serum and plasma for effective TDM and (2) for TDM, serum or plasma may be used.

Beta-amyloid 1-42 (Aβ42) and tau protein are cerebral spinal fluid (CSF) biomarkers for progression to AD. The sensitivity requirements for Aβ42 and total tau assays are in the pg/mL range, a challenge for any methodology. The use of these biomarkers for clinical purposes requires assays that can reliably deliver accurate and reproducible results. New prototype immunoassays for Aβ42 and total tau on a fully automated clinical analyzer [Siemens Healthineers ADVIA Centaur® XPT Immunoassay System] are under development to enable routine testing of AD biomarkers in clinical laboratories. Each assay uses an antigen/antibody sandwich format and chemiluminescence detection. Standard curves from 0 to 4000 pg/mL and 0 to 6500 pg/mL were generated for the Aβ42 and Tau assays, respectively. Synthetic Aβ42 and recombinant tau-441 were spiked into an immunodepleted (ID) CSF pool and tested. Precision was evaluated over 6 days by testing a CSF pool and buffer controls on two analyzers. Functional sensitivity was estimated using the precision data. Clinical utility was evaluated by testing 30 clinical CSF samples from subjects with and without AD. Recoveries of spiked Aβ42 from CSF ranged from -13% to +0.7% over 113 to 3179 pg/mL. Recoveries of spiked tau-441 from CSF ranged from -11% to +13% over 35 to 3826 pg/mL. The repeatability and within-laboratory precision of the Aβ42 assay ranged from 1.3% to 8.6% and 1.9% to 9.9%, respectively, over 107 to 2005 pg/mL. The repeatability and within-laboratory precision of the total tau assay ranged from 1.2% to 10.8% and 1.6% to 12.5%, respectively, over 108 to 3300 pg/mL. The functional sensitivities of the Aβ42 and total tau assays were estimated to be < 200 pg/mL and < 80 pg/mL, respectively. Using ROC analysis, a ratio of Aβ42 and total tau results was able to differentiate AD from non-AD subjects with 90% sensitivity and 95% specificity (AUC 0.95). When finalized, the fully automated Aβ42 and total tau assays will have performance characteristics suitable for routine use in a clinical laboratory. The performance of the assays in combination with automation will make testing for these important biomarkers more reliable and routine.

measure clozapine levels in serum and plasma, were modified for testing clozapine levels in whole blood (WB) with a POCT analyser, the MyCare Insite. This small, portable device generated clozapine levels in under 7 minutes for near patient testing. As reported in the literature, WB contains ~20% less clozapine than serum or plasma prepared from WB.21,22 Clozapine monitoring recommendations are based on studies on serum/plasma levels; the WB results were standardized to give results comparable to serum measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Reassignment of calibrator concentrations was performed with a “training set” of 95 clozapine blood samples collected from 21 patients. Capillary WB and EDTA plasma were simultaneously collected and clozapine was measured on the MyCare Insite, and by LC-MS/MS. Performance validation was done according to Clinical Laboratory Standards Institute Guidelines for precision, interference, linearity, detection capability and method comparison. Each study was conducted with at least five operators and fifteen analysers. Samples for the method comparison were collected during patients’ regular visits at two sites of the South London and Maudsley NHS Foundation Trust. Capillary whole blood samples were tested immediately using the MyCare Insite Clozapine Test by two operators using four analysers. Matching venous plasma samples were tested by a validated LC-MS/MS method. Repeatability (n=2) was performed with six individual patients.

Background: Gemcitabine (2′,2′-difluoro-2′-deoxycytidine) is a nucleoside analog used as a single agent and in combination regimens for the treatment of a variety of solid tumors. Several studies have shown a relationship between gemcitabine peak plasma concentration (C max ) and hematological toxicity. An immunoassay for gemcitabine in plasma was developed and validated to facilitate therapeutic drug monitoring (TDM) by providing an economical, robust method for automated chemistry analyzers. Methods: A monoclonal antibody was coated on nanoparticles to develop a homogenous agglutination inhibition assay. To prevent ex vivo degradation of gemcitabine in blood, tetrahydrouridine was used as a sample stabilizer. Validation was conducted for precision, recovery, cross-reactivity, and linearity on a Beckman Coulter AU480. Verification was performed on an AU5800 in a hospital laboratory. A method comparison was performed with (LC-MS/MS) liquid chromatography tandem mass spectrometry using clinical samples. Selectivity was demonstrated by testing cross-reactivity of the major metabolite, 2′,2′-difluorodeoxyuridine. Results: Coefficients of variation for repeatability and within-laboratory precision were <8%. The deviation between measured and assigned values was <3%. Linear range was from 0.40 to 33.02 μ/mL (1.5–125.5 μM). Correlation with validated LC-MS/MS methods was R 2 = 0.977. The assay was specific for gemcitabine: there was no cross-reactivity to 2′,2′-difluorodeoxyuridine, chemotherapeutics, concomitant, or common medications tested. Tetrahydrouridine was packaged in single-use syringes. Gemcitabine stability in whole blood was extended to 8 hours (at room temperature) and in plasma to 8 days (2–8°C). Conclusions: The assay demonstrated the selectivity, test range, precision, and linearity to perform reliable measurements of gemcitabine in plasma. The addition of stabilizer improved the sample handling. Using general clinical chemistry analyzers, gemcitabine could be measured for TDM.

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 mu L 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.

Background: Measurement of parathyroid hormone (PTH) is important for the clinical assessment of parathyroid disease and calcium homeostasis. Previous studies have demonstrated decreased stability of PTH in serum versus plasma specimens, although the precise mechanism for this difference has not been established. If thrombin activation during clot formation is responsible, the effect should be exacerbated in tubes containing additional thrombin. Exogenous thrombin is a constituent of Vacutainer Rapid Serum TubesTM (RST; BD Diagnostics, Franklin Lakes, NJ), which include bovine thrombin to induce accelerated clot formation. As a known thrombin cleavage site exists in the 84 amino acid human PTH polypeptide, we hypothesized that ex vivo PTH cleavage in RST tubes might occur. Such a possibility has analytical and clinical implications, as intact PTH diagnostic tests are sandwich immunoassays that use paired capture/detection antibodies to the Nand C-terminal regions of PTH. Methods: 1) Screening Study: Initial experiments were conducted to determine whether measurement of analytes with potential thrombin cleavage sites would be affected by RST tubes. Aliquots from previously collected serum specimens were incubated for 24 hrs in either an RST or a Serum Separator TubeTM (SST) before analysis on a cobas e602 immunoanalyzer (Roche Diagnostics, Indianapolis, IN). A subset of replicate experiments was performed on an ARCHITECT i1000SR (Abbott Diagnostics, Lake Forest, IL). 2) Fresh Collections: To verify fi ndings in freshly collected specimens, three tubes of fresh blood were drawn from each of 10 healthy normal donors. Specifi cally, we collected one Plasma Separator TubeTM (PST), one SST, and one RST from each donor. After clotting and centrifugation, intact PTH assays on the cobas e602 were performed at multiple time points on aliquots held at ambient temperature or 4°C. Confi rmatory experiments were conducted using exogenous thrombin and the direct thrombin inhibitor hirudin. Results: In screening studies, PTH results were lower after specimen incubation in RSTs versus SSTs. These fi ndings were confi rmed in additional time-course experiments and on a separate immunoassay platform. In fresh collections, RST and SST specimens stored at room temperature also showed a decrease in PTH results (versus PST specimens) beginning at our earliest measurement time-point (approximately 1 hr post-collection). The magnitude of this decrease was more prominent in RSTs versus SSTs. A similar (but smaller and slower) decrease in PTH results in serum specimens was seen in aliquots stored at 4°C. Further studies confi rmed that the decrease in measured PTH was thrombin-mediated, as it was blocked by hirudin. Conclusion: The present study demonstrates that thrombin is responsible for the decrease in PTH results observed in RSTs. It is presumed that endogenous human thrombin, activated during clot formation, may be responsible for the smaller decreases observed in SSTs. As there is an incomplete understanding of which additional polypeptides may possess bovine thrombin cleavage sites, these studies provide a simplifi ed screening strategy that laboratories can use when evaluating RSTs for assays at their own institutions.

Evaluation of cerebrospinal fluid (CSF) biomarkers in Alzheimer's disease (AD) is becoming increasingly important to improve the reliability of ante-mortem disease diagnosis to ensure proper patient management. Development of highly precise assays for amyloid 1-42 (Aβ42) and tau protein has allowed investigators to better characterize pre-analytical sample handling factors that may affect clinical interpretation. A prospective collection study was designed to model different CSF handling scenarios for storage conditions at the clinical site, shipping, and storage and handling at the testing site. CSF was prospectively collected from 46 healthy individuals under an IRB approved protocol. A 10mL CSF aliquot was collected by lumbar puncture in the L3/L4 or L4/L5 interstitial space. One milliliter aliquots were stored and shipped at: -80°C, -20°C, 4°C and 25°C. This study investigated storage, shipping and handling conditions, including freeze/thaws for 6 randomly selected patients using an immunometic chemiluminescent assay. Additional studies examined the influence of storage tube material. Compared to -80°C, aliquots stored at -20°C and 25°C had Aβ42 losses from 5 to 20% and 40% loss for aliquots stored at ambient temperatures for 48 hours. There was little to no effect on tau concentrations across the range of storage temperatures. Aliquots initially stored at -80°C or ambient temperature showed the greatest decreases in measured Aβ42 levels (10 to 15%) following a second freeze/thaw cycle, whereas no loss of tau was observed with a freeze/thaw cycle. Thawing CSF samples at ambient temperature versus a water bath did not affect concentrations of either biomarker. Results were consistent between patients. Storing samples for 2h at ambient temperature post thaw resulted in less than 10% loss for either marker. The Eppendorf Lobind® tubes allowed transfer of CSF and storage at 4°C for up to six days without significant loss of either Aβ42 or tau biomarkers. Storage and transfer conditions of CSF can have a significant effect on the level of Aβ42 measured but do not impact tau concentrations appreciably. The reliability and utility of these markers in routine testing can be improved by minimizing analyte losses by choice of conditions from collection to testing site.

Tau concentration in cerebrospinal fluid (CSF) has been shown to correlate with Alzheimer's disease (AD) progression and is a key measurement in the identification of people at risk for progressing to Alzheimer's disease (AD) and the proper management of people with AD. The use of such markers for clinical purposes requires assays that can deliver consistent analytical and clinical performance. We report on the VITROS ® Immunodiagnostic Products Tau Assay 1 assay under development for the measurement of tau in CSF on the VITROS Immunodiagnostic Systems. The objective of this study was to demonstrate consistent performance of the assay across lots and across instrument platforms. Two lots of VITROS Tau assay reagent were tested on the VITROS ® ECiQ and VITROS ® 3600 Immunodiagnostic Systems to evaluate lot-to-lot and system-to-system variability. Fifty individual CSF samples and controls were run in singleton with controls run at the beginning and end of each test. The results from the VITROS 3600 using Lot 1 reagent were used as the control condition and linear regression was performed, as well as the %CV calculated across all runs. The system-to-system comparison (Lot 1 - ECiQ vs. Lot 1 - 3600) slope, intercept, and correlation (r) results were 0.9913, 2.87, and 1.000 respectively. The lot-to-lot comparison (Lot 2 - 3600 vs. Lot 1 - 3600) slope, intercept, and correlation (r) results were 0.979, -8.70, and 0.999 respectively. Across lots and systems (Lot 2 - ECiQ vs. Lot 1 - 3600) slope, intercept, and correlation (r) results were 0.981, 0.65, and 1.000 respectively. The mean doses of the controls (n = 8 per control) ranged from 64.8 - 4145 pg/mL with an overall %CV from 0.9% - 4.1%. The mean doses of the individual CSF samples (n = 4 per CSF) ranged from 56.2 - 1412 pg/mL with a %CV from 0.5% - 5.8%, overall 2.4%CV across lots and systems. The VITROS Immunodiagnostic Products Tau Assay 1 has demonstrated consistent results across reagent lots and across systems.

Tau concentration in CSF has been shown to correlate with Alzheimer's disease (AD) progression and is a key measurement in the assessment of AD risk and status. The use of such markers for clinical purposes requires assays that can deliver consistent analytical and clinical performance. We report on the VITROS ® Immunodiagnostic Products tau assay for the measurement of tau in CSF on the VITROS Immunodiagnostic Systems. The objective of this study was to demonstrate the stability of the assay reagents over a 29 week period. One lot of VITROS Tau assay reagent was stored at 2–8°C and tested at 4 timepoints over a 29 week period to demonstrate the stability of the reagents. At each timepoint, the reagents were calibrated and used to test two CSF pools containing approximately 400 and 2700pg/mL of endogenous tau. The percent change in the measured tau level for each CSF pool at each timepoint was calculated. The %CV was also calculated across all timepoints for both CSF pools. For the 29 week study, the mean percent differences in measured CSF tau levels vs. time zero were -3.7% and -2.0% for the low and high CSF pools, respectively. The individual percent differences from time zero for both CSF pools across all timepoints ranged from -8.1% to -0.6%. The pg/mL %CV's across all timepoints were 3.8% and 1.6% for the low and high CSF pools, respectively. The VITROS Immunodiagnostic Products tau assay has demonstrated consistent results with reagents stored at 2–8°C for over 6 months.

Beta amyloid 1–42 (Aβ42) concentration in cerebrospinal fluid (CSF) has been shown to correlate with Alzheimer's disease (AD) progression and is a key measurement in identification of people at risk for progressing to AD and the proper management of people with AD. The use of such markers for clinical purposes requires assays that can deliver consistent analytical and clinical performance. We report on the VITROS® Immunodiagnostic Products Amyloid Beta 42 Assay (AB-42)1 under development for measurement of Aβ42 in CSF on VITROS Immunodiagnostic Systems. The objective of this study was to demonstrate consistent performance of the assay across lots and instrument platforms. Two lots of VITROS AB-42 assay reagent were tested on the VITROS® ECiQ and VITROS® 3600 Immunodiagnostic Systems to evaluate lot-to-lot and system-to-system variability. Fifty individual CSF samples and controls were run in singleton with controls run at the beginning and end of each test. The results from the VITROS 3600 using Lot 1 reagent were used as the control condition and linear regression was performed, as well as the %CV calculated across all runs. The system-to-system comparison (Lot 1 - ECiQ vs. Lot 1 - 3600) slope, intercept, and correlation (r) results were 1.001, 14.0, and 0.998 respectively. The lot-to-lot comparison (Lot 2 - 3600 vs. Lot 1 - 3600) slope, intercept, and correlation (r) results were 0.992, -5.69, and 0.999 respectively. Across lots and systems (Lot 2 - ECiQ vs. Lot 1 - 3600) slope, intercept, and correlation (r) results were 1.014, -0.84, and 0.998 respectively. The mean doses of the controls (n = 8 per control) ranged from 55.0 - 1899 pg/mL with an overall %CV from 1.6% - 5.0%. The mean doses of the individual CSF samples (n = 4 per CSF) ranged from 41.8 - 1236 pg/mL with an overall %CV from 1.0% - 25.3%. Only one CSF sample with a concentration >100 pg/mL had a CV >10%. The VITROS Immunodiagnostic Products Amyloid Beta 42 Assay 1 has demonstrated consistent results across reagent lots and across systems.

Evaluation of cerebrospinal fluid (CSF) biomarkers in Alzheimer's disease (AD) is increasingly more important for improving the probability of ante-mortem diagnosis of AD, ensuring proper patient management. Use of such markers for clinical purposes, in conjunction with potential disease-modifying therapies requires assays that can deliver high analytical and clinical performance. Two biomarkers, beta amyloid 1–42 (Aβ42) and tau protein, correlate with disease progression and a number of research use only (RUO) tests are available. The VITROS® Immunodiagnostic Products Tau Assay 1 is an accurate and robust assay under development for tau that shows a marked improvement in precision, linearity and reproducibility when compared to currently available tests. An in vitro diagnostic test for tau is being developed for the VITROS Immunodiagnostic Systems, and analytical performance was evaluated following CLSI guidelines using two VITROS Tau assay reagent lots, 3 CSF pools, and 3 controls. Linearity was tested with 11 admixtures of endogenous tau in CSF and recombinant tau 441 in buffer. Interference testing included commonly prescribed drugs, recombinant tau, Aβ peptides, and endogenous substances. Limit of detection (LoD) and lower limit of quantitation were confirmed with 20 replicates per day on 3 days. The median within-laboratory coefficient of variation (CV) for the 3 CSF pools was 3.3% with a range of 2.4% 4.2%. The controls' within-laboratory CV was ≤1.2%. Repeatability CVs were ≤1.9% for CSF pools and ≤1.2% for controls. The assay showed good linearity across the measuring range (0–8,000pg/mL) with the admixture of endogenous and synthetic peptide. Bias introduced by commonly prescribed drugs at high levels was <±10%. The interference from Aβ peptides at greater than known physiological concentrations as well as from endogenous substances was <5%. The limit of detection was 9.5pg/mL; the lower limit of quantitation was 40pg/mL. VITROS Immunodiagnostic Products Tau Assay 1 demonstrates excellent analytical performance: it is precise, linear and the tested interferences cause no significant bias. The fully automated format and ability to perform these assays in a clinical laboratory should make testing for these important markers more reliable and routine.

Beta amyloid 1–42 (Aβ42) concentration in CSF has been shown to correlate with Alzheimer's disease (AD) progression and is a key measurement in the assessment of AD risk and status. The use of such markers for clinical purposes requires assays that can deliver consistent analytical and clinical performance. We report on the VITROS ® Immunodiagnostic Products Amyloid Beta 42 Assay (AB-42)1 under development for the measurement of Aβ42 in CSF on the VITROS Immunodiagnostic Systems. The objective of this study was to demonstrate the stability of the assay reagents over a 54 week period. One lot of VITROS AB-42 assay reagent was stored at 2–8°C and tested on the VITROS ® ECiQ Immunodiagnostic System at 13 timepoints over a 54 week period to demonstrate the stability of the reagents. Five controls ranging in concentration from50 pg/mL to 1200 pg/mL were run in duplicate at each timepoint. In the first part of the study the reagents were calibrated off Reference Calibrators every 28 days over a 16 week period to demonstrate calibration stability. In the second part of the study the reagents were calibrated at each timepoint over a 54 week period. In each study the percent change in predicted concentration at each timepoint was calculated along with the mean and range in percent change. The %CV was also calculated across all timepoints in each study. For the 16 week study, the mean percent difference from time zero ranged from -1.3% to 0.9% across all controls. The percent difference from time zero at each timepoint ranged from -5.5% to 3.2%. The %CV across all timepoints for each control ranged from 1.1% to 1.9%. For the 54 week study, the mean percent difference from time zero ranged from 0.3% to 2.3% across all controls. The percent difference from time zero at each timepoint ranged from -1.3% to 4.5%. The %CV across all timepoints for each control ranged from 1.0% to 1.5%. The VITROS Immunodiagnostic Products Amyloid Beta 42 Assay 1 has demonstrated consistent results with reagents stored at 2–8°C over more than a year.

Beta amyloid 1–42 (Aβ42) and tau assays are under development for the VITROS Immunodiagnostic Systems. These cerebrospinal fluid (CSF) biomarkers have previously been demonstrated to correlate with risk of progression from cognitively normal (CN) to Alzheimer's disease (AD). Identification of at-risk patients earlier in the development and progression of disease is critical for therapeutic development and disease management. Appropriate assay cut-points are vital for the accurate determination of risk of disease progression. This study was conducted to evaluate the performance of preliminary cut-points for the VITROS® Immunodiagnostic Products Amyloid Beta 42 Assay (AB-42)1 and Tau Assay 1. Banked samples were obtained from an expert center with a defined protocol for collection and MCI classification. Diagnostic status was defined by clinical assessment for CN (n=30), AD (n=30), MCI-who remained dementia free (MCI-s; n=30) or MCI-who progressed to AD dementia (MCI-p; n=30). Samples were measured concurrently with the VITROS AB-42 and VITROS Tau assays and a commercial RUO ELISA. Receiver operator curve (ROC) analysis was used to analyze cut-points and assess sensitivity and specificity. The VITROS/RUO method comparison correlation coefficients (R) were >0.97. Sensitivity and specificity obtained in the AD-CN population was 93.3%&83.3% for VITROS AB-42; 76.7%&96.7% for VITROS Tau; 96.7%&93.3% for the VITROS Tau/AB-42 ratio based on the preliminary cut-points. For MCI-p & MCI-s classified samples, sensitivity and specificity were 76.7%&93.3% for VITROS AB-42; 83.3%&73.3% for VITROS Tau; 76.7%&90.0% for the ratio. In the MCI-p &CN population the sensitivity and specificity calculated were 76.7%&93.3% for VITROS AB-42; 83.3%&80.0% for VITROS Tau; 100%&83.3% for the ratio. The area under the curve (AUC) for the VITROS Tau:AB-42 ratio was 0.992 for AD&CN, 0.893 for MCI-p&MCI-s, and 0.954 for MCI-p&CN. The Aβ42 ROC correlation was 0.98. The VITROS Tau and VITROS Tau/AB-42 ratio tests were not statistically different from the RUO tests. A common cut-point for both populations produced observed sensitivity and specificity of 77%& 90% for MCI-p&MCI. For AD&CN observed sensitivity and specificity were 87% and 97%. The VITROS Immunodiagnostic Products Amyloid Beta 42 Assay 1 and Tau Assay 1 discriminated between MCI-p&MCI-s and AD&CN populations with acceptable specificity and sensitivity using preliminary cut-points.

Evaluation of cerebrospinal fluid (CSF) biomarkers in Alzheimer's disease (AD) is becoming increasingly important to improve the reliability of ante-mortem disease diagnosis to ensure proper patient management. Two biomarkers, beta amyloid 1–42 (Aβ42) and Tau, have been shown to correlate with disease progression, and a number of research use only (RUO) tests are available. Assays delivering excellent analytical and clinical performance are required to use these markers for clinical purposes in conjunction with potential disease-modifying therapies. This study evaluated the consistency of performance of the VITROS® Immunodiagnostic Products Amyloid Beta 42(AB-42)1 and Tau 1 assays over multiple reagent lots using CSF specimens from AD patients. Assays for Aβ42 and tau are being developed on the VITROS Immunodiagnostic Systems. Performance was evaluated using two reagent lots. Twelve CSF samples were tested in duplicate for five days with both lots. To test diagnostic efficiency, clinical samples from 30 clinically normal and 30 AD patients were tested in the VITROS Tau, VITROS AB-42 and RUO assays. The differences in the mean tau concentrations between lot 2 and lot 1 for each of the 12 CSF samples ranged from 0.8% to 2.2% in the precision study. For VITROS AB-42 lot 2 vs. lot 1 mean concentration variations were between -0.6% and 1.7%. Mean values were between 319–1303pg/mL for tau and 292–588pg/mL for Aβ42. Repeatability coefficients of variation (CV) were 0.4%-2.0% with two lots of VITROS Tau reagents and 0.7% and 3.6% for the VITROS AB-42 lots. Within-laboratory CVs were 1.3%-3.0% for VITROS Tau and 2.7%-6.4% for VITROS AB-42. The VITROS Tau:AB-42 ratio ROC maximum efficiency point was 98.3%. The VITROS Tau:AB-42 ratio resulted in 100% sensitivity and 96.7% specificity. Using the same cohort, maximum efficiency for the RUO CSF tau and Aβ42assays was 80%, and 98.3% and 96.7% for tau: Aβ42. The VITROS Immunodiagnostic Products Amyloid Beta 42 Assay 1 and Tau Assay 1 have demonstrated good precision over two lots of reagents and shown good discrimination between AD and control patients necessary for the effective use of Aβ42 and tau measurements in clinical practice.

563 Background: 5-fluorouracil (5-FU) is one of the most widely used chemotherapy agents in the treatment of colorectal cancer. Studies over the last 30 years have demonstrated wide pharmacokinetic variability of 5-FU which can lead to undue toxicity and suboptimal treatment. Recent clinical studies have demonstrated that managing plasma 5-FU levels and adjusting doses to target steady state concentrations effectively minimizes toxicity and improves outcome. Methods: An existing immunoassay using a novel antibody to 5-FU was modified to directly quantify 5-FU in patient plasma samples without any sample pretreatment. The assay was adapted to Beckman Coulter AU analyzers and to Roche COBAS c 111 analyzer. Method comparison to LC-MS/MS, limit of detection (LoD), lower limit of quantitation (LLoQ), precision, and linearity of the assay were evaluated. The assay was also validated by testing samples of patients being treated by modified FOLFOX6 from an on-going clinical trial ( NCT00943137 ). Results: The modified immunoassay was confirmed to have a linear reportable range from 85 to 18,000ng/mL and a LoD of 52ng/mL. The immunoassay was established to have good precision (CV<6%) around the medical decision points for FOLFOX and FOLFIRI regimens. Method comparison of the immunoassay obtained by testing 58 clinical samples from patients under 5-FU treatment provides excellent correlation to LC- MS/MS: Deming slope 1 ± 0.05, R > 0.98. Another 82 samples of patients on modified FOLFOX6 regimen from clinical trial (( NCT00943137 ) were tested using the immunoassay and range of these sample were reported to be from 95 to 2,970 ng/mL. Conclusions: The assay provided the performance required to rapidly quantify 5-FU plasma concentrations. It was fast (time to 1st result < 10 minutes), precise, correlated well to a physical method, required only 100uL sample on the analyzer and 7ul for actual testing per assay, and was easily adapted to a variety of clinical analyzers. Clinical and pharmacokinetic laboratories could use this assay to introduce an evidence-based approach to optimize 5-FU dosing that would have higher throughput, simpler methodology, and require less labor, space or expense than the physical methods. [Table: see text]

Background: Busulfan (BU), a bifunctional alkylating agent, is the most widely used chemotherapeutic agent as a component of high-dose conditioning regimens for hematopoietic stem cell transplantation (HSCT). Studies have shown that maintaining a targeted exposure [expressed as area under the time-curve (AUC)] throughout the complete regimen is essential for successful engraftment, reduced risk of graft vs. host disease or venoocclusive disease. For optimal therapeutic drug management (TDM), an assay that can deliver rapid high quality results would be desirable. Methods: Monoclonal antibodies bound to nanoparticles were used to develop an immunoassay for the Roche c111 chemistry analyzer. The assay was monitored at 629 nm and quantitated with a stabilized calibrator of busulfan. Assay precision, linearity, calibration stability and limit of detection (LoD) were determined. Clinical samples were analyzed and correlated with GC-MS. Results: With six calibrators from 0–2,000 ng/mL, a stabilized busulfan derivative, and auto-dilution, plasma samples with concentrations up to 10,000 ng/mL could be determined. Time-to-first-result was 12 min and 25 samples/h could be measured from a stored standard curve. Imprecision across the range of assay was <7% and the assay was linear upon dilution over the entire range. Cross-reactivities for sulfolane, tetrathiophene and 3-hydroxsulfolane were <1%. The LoD was 50 ng/mL with a functional sensitivity of 100 ng/mL. Assay results of clinical samples (237 to 1,711ng/mL) correlated well with GC-MS results: (R > 0.98, slope 1.05, intercept = 39 ng/mL). Conclusions: This immunoassay is suitable for determining busulfan concentrations in plasma. It offers advantages of turn-around time, small sample size, no sample pre-treatment and a stable calibrator that can be stored at room temperature. Automation and rapid turn-around time with a small benchtop analyzer enable efficient routine monitoring of busulfan concentrations in clinical practice which can lead to better patient care.