Enzyme Multiplied Immunoassay Technique Assay Research Articles

LC-MS/MS and EMIT measure the whole blood concentration of cyclosporine A: The two methods yield concordant results within the dynamic range of the latter, but the former shows broader application scenarios

Cyclosporine A (CsA) is a widely used immunosuppressive drug with a narrow therapeutic index and large individual differences. Its therapeutic and toxic effects are closely related to blood drug concentrations, requiring routine therapeutic drug monitoring (TDM). The current main methods for TDM of CsA are enzyme multiplied immunoassay technique (EMIT) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, few study on the method comparison of the EMIT and LC-MS/MS for the measurement of whole blood CsA concentration in children has been reported. In this study, we developed a simple and sensitive LC-MS/MS assay for the determination of CsA, and 657 cases of CsA concentrations were determined from 197 pediatric patients by a routine EMIT assay and by the validated in-house LC-MS/MS method on the same batch of samples, aimed to address the aforementioned concern. Consistency between the two assays was evaluated using linear regression and Bland-Altman analysis. The linear range of LC-MS/MS was 0.500–2000 ng/mL and that of the EMIT was 40–500 ng/mL, respectively. Overall, the correlation between the two methods was significant (r-value ranging from 0.8842 to 0.9441). Unsatisfactory consistency was observed in the concentrations < 40 ng/mL (r = 0.7325) and 200–500 ng/mL (r = 0.6851). Bland-Altman plot showed a mean bias of −18.0 % (±1.96 SD, −73.8 to 37.8 %) between EMIT and LC-MS/MS. For Passing-Bablok regression between EMIT and LC-MS/MS did not differ significantly (p > 0.05). In conclusion, the two methods were closely correlated, but the CsA concentration by LC-MS/MS assay was slightly higher than that by EMIT method. Switching from the EMIT assay to the LC-MS/MS method was acceptable, and the LC-MS/MS method will receive broader application in clinical settings due to its better analytical capabilities, but the results need to be further verified in different laboratories.

Comparison of LC-MS/MS and EMIT methods for the precise determination of blood sirolimus in children with vascular anomalies.

Sirolimus (SRL) is a mammalian target of rapamycin (mTOR) inhibitor. The whole blood concentration of SRL is routinely monitored to tailor dosage and prevent toxicity. Currently, the enzyme multiplied immunoassay technique (EMIT) is often applied to perform therapeutic drug monitoring (TDM) of SRL, but the cross-reactivity with various metabolites is of great concern. A more specific method is required, such as liquid chromatography–tandem mass spectrometry (LC-MS/MS). However, no study on the method comparison of the EMIT and LC-MS/MS for the measurement of whole blood SRL concentration in children with vascular anomalies has been reported. This study developed a simple and sensitive LC-MS/MS assay for the determination of SRL. Meanwhile, consistency between LC-MS/MS and the EMIT was evaluated by linear regression and Bland–Altman analysis. Whole blood samples were deproteinized with methanol for erythrocyte lysis, and the resulting solution was injected into the LC-MS/MS system using the positive electrospray ionization mode. The multiple reaction monitoring transitions of m/z 931.7 → 864.6 and m/z 934.7 → 864.6 were used for SRL and SRL-d3 as the internal standards, respectively. The analytes were separated on a C18 column with a gradient mobile phase (0.1 mM formic acid and 0.05 mM ammonium acetate in methanol/ultrapure water). Blood samples collected from children with vascular anomalies undergoing SRL therapy were tested by EMIT and by LC-MS/MS. The linear range of LC-MS/MS was 0.500–50.0 ng/ml and that of the EMIT was 3.50–30.0 ng/ml. A significant positive correlation between the two assays was established with a regression equation described as [ EMIT ] = 1.281 × [ LC−MS/MS ] + 2.450 (r = 0.8361). Bland–Altman plots showed a mean concentration overestimation of 4.7 ng/ml [95% CI: (−3.1, 12.6)] and a positive bias of 63.1% [95% CI: (−36.1, 162.3)] generated by the EMIT more than that of by LC-MS/MS. In conclusion, the two methods were closely correlated, indicating that switching between the two methods is feasible. Considering the overestimation nature of the EMIT assay, switching from the EMIT to the LC-MS/MS method deserves close attention and necessary re-evaluation for the target therapeutic reference range, may be required when methods are switched within the same clinical laboratory or results are compared between different laboratories.

Switching Between LC-ESI-MS/MS and EMIT Methods for Routine TDM of Valproic Acid in Pediatric Patients With Epilepsy: What Clinicians and Researchers Need to Know.

Background: Valproic acid (VPA) is a widely used antiseizure medication and its dosing needs to be tailored individually through therapeutic drug monitoring (TDM) to avoid or prevent toxicity. Currently, immune-enzymatic assays such as Enzyme Multiplied Immunoassay Technique (EMIT), and Liquid Chromatography (LC)-based techniques, particularly coupled to Electrospray Ionization Tandem Mass Spectrometry (LC–ESI-MS/MS), resulting a potential lack of concordance between laboratories. Methods: In this study, plasma VPA concentrations were determined for 711 pediatric patients with epilepsy by a routine EMIT assay and by a validated in-house LC-ESI-MS/MS method on the same group of samples, aimed to address the aforementioned concern. Consistency between two assays was evaluated using linear regression and Bland-Altman analysis. Results: The calibration curve was linear in the range of 5.00–300 μg/ml for LC-ESI-MS/MS method and 1.00–150 μg/ml for EMIT assay, respectively. The two methods were proven to be accurate with quality control samples. As a result, a significant correlation between two methods was obtained with a regression equation described as (r 2 = 0.9281). Bland-Altman plot showed a mean bias of 14.5 μg/ml (95% confidence interval (CI) (−0.2, 29.2) and a mean increase of 27.8% (95% CI (3.3, 52.4) measured by EMIT assay more than that measured by LC-ESI-MS/MS method. Conclusion: In conclusion, two methods were closely correlated, but EMIT assay overestimate VPA levels in human plasma compared with LC-ESI-MS/MS method. Due to the observed significant discordance between the tested methods, switching from immunoassays to LC-based techniques for TDM of VPA deserves close attention and therapeutic range of 35.0–75.0 μg/ml may be feasible. However, further studies are needed to evaluate the eligibility of this alternative range in the clinical practice. Clinicians should be informed when switching the VPA quantitation methods during the clinical practice.

Retrospective evaluation and prediction of clearance and toxicity of high dose methotrexate in childhood acute lymphoblastic leukemia patients

To find the predictors of High Dose Methotrexate toxicities in childhood Acute Lymphoblastic Leukemia Patients. This study included 198 Childhood Acute Lymphoblastic Leukemia patients (303 infusions) who were treated with High Dose Methotrexate. Methotrexate levels at different time point were measured by modified enzyme multiplied immunoassay technique assay. The correlation between Methotrexate levels and toxicity was evaluated by Receiver Operating Characteristic curve. When the Methotrexate level at 42 h was lower than 0.76 µmol/L, the sensitivity for predicting thorough clearance at 66 h was 90.78%. When the Methotrexate level at 42 h was higher than1.5 µmol/L, the sensitivity for predicting delayed clearance was 82.17%. When the Methotrexate level at 66 h was higher than 0.5 µmol/L, the sensitivity for predicting Methotrexate toxicity was 89.09%. When the Methotrexate level at 66 h was lower than 0.1 µmol/L, the sensitivity for predicting Methotrexate nontoxicity was 92.73%. The Methotrexate level at 42 h could be predictor for delayed clearance. The Methotrexate level at 66 h could be predictor for toxicity.

Modified enzyme multiplied immunoassay technique of methotrexate assay to improve sensitivity and reduce cost

BackgroundMethotrexate is an important component in many chemotherapy protocols. The blood concentration of Methotrexate is used to determine the regimen of folinic acid. However, the lower limit of Siemens assay kit is 0.30 μmol/L in China. This study extended the limit from 0.3 to 0.05 μmol/L and reduced the test cost by optimizing the parameters of Enzyme Multiplied Immunoassay Technique assay.MethodsParameters of Enzyme Multiplied Immunoassay Technique assay were modified to decrease the volume of reagents A and B. Then a standard curve with a new custom set of calibrators was prepared to detect low concentration. Intra-day and inter-day imprecision were assessed by control material and samples. The linearity of the modified assay was verified by analyzing a range of quality controls with known concentration from 0.05 to 1.00 μmol/L. At last, the same samples were tested by modified Enzyme Multiplied Immunoassay Technique assay and Liquid Chromatography-tandem Mass Spectrometry assay respectively. A simple linear regression was performed to verify the validity of the modified Enzyme Multiplied Immunoassay Technique assay.ResultsIntra-day and inter-day imprecision show good reproducibility at all levels (0.05, 0.12, 0.43, 0.82 μmol/L). The linearity equation of modified assay was y = 0.9913x + 0.0046, in which y was the mean of measured concentration and x was the target concentration (R2 = 0.9994). In the range of 0.05–10.00 μmol/L, correlation between the Modified assay and Liquid Chromatography-tandem Mass Spectrometry assay was significant (r = 0.9968). In the range of 0.30–10.00 μmol/L, the correlation between modified and commercial assays was significant (r = 0.9987) as well.ConclusionsThe modified assay enhanced the sensitivity of Siemens VIVA-E to 0.05 μmol/L. In addition, the test number of a reagent Kit increased from 140 to 210. This means the cost of detection was reduced about 30%.

Inter-laboratory Variability of Current Immunoassay Methods for Tacrolimus among Japanese Hospitals.

The aim of this study was to assess inter-hospital laboratory variability (coefficient of variation; CV) of immunoassay methods for tacrolimus and the comparability of control samples and results obtained by immunoassay measurements. One hundred seven hospital laboratories routinely performing therapeutic drug monitoring (TDM) of tacrolimus participated in the study. Thirteen spiked samples with known tacrolimus concentrations in the range of 0-26.0 ng/mL were prepared. Each spiked sample was analyzed according to the manufacturer's instructions using an affinity column-mediated immunoassay (ACMIA) on a Dimension(®) analyzer, the enzyme multiplied immunoassay technique (EMIT) on a Viva-E(®) analyzer, a chemiluminescent enzyme immunoassay (CLIA) on the Architect(®) system, and the electro-chemiluminescence immunoassay (ECLIA) on a cobas(®) analyzer. The 20% coefficient of variation values for the CLIA, ACMIA, EMIT, and ECLIA assays in the hospital laboratories were 1.82, 5.36, 4.59, and 0.89 ng/mL, respectively. CLIA and ECLIA had positive biases at concentrations of tacrolimus above 12 ng/mL relative to the spiked concentration, whereas the assay bias for ACMIA tended to be more negative at concentrations of tacrolimus above 6 ng/mL. EMIT had positive biases over the wide concentration range of 0.0-26.0 ng/mL (mean of mean errors 1.224). CLIA and ECLIA provided adequate precision at the target tacrolimus concentration of 3.0 ng/mL, whereas ACMIA and EMIT were unable to respond to target concentrations between 3.0 and 5.0 ng/mL for renal transplant recipients. Appropriate assessment of tacrolimus concentration by an assay having higher sensitivity, precision, and accuracy is necessary to ensure long-term survival of transplant recipients receiving tacrolimus.

Improved Sensitivity for Methotrexate Analysis Using Enzyme Multiplied Immunoassay Technique on the Siemens Viva-E Instrument

The available assay kit for methotrexate (MTX) using the Syva enzyme multiplied immunoassay technique (EMIT) reagents on the Siemens Viva-E instrument allows for the detection of MTX in serum or plasma to concentrations as low as 0.3 μmole/L. Current clinical decision points for MTX therapeutic drug monitoring and leucorvorin rescue exist at concentrations below that limit. The goal of this study was to lower the limit of MTX quantitation to 0.05 μmole/L using the EMIT assay technology. EMIT MTX assay parameters were modified on the Viva-E instrument to increase the sample volume, alter the calibration method, and employ an alternate calibrator set created to achieve lower detection. Intraassay and interassay precision was assessed for MTX controls. We observed a CV of 9.4% for intraassay precision with a bias of <0.01% and a CV of 15.7% for interassay precision with a bias of 22.5% for the 0.05 μmole/L control. Precision data for all other controls were <4%. The modified EMIT MTX assay and the unmodified approved assay were compared with a high sensitivity fluorescence polarization immunoassay method. Linear regression of correlation data revealed that both the modified and the commercial EMIT assays produced positive bias compared with the high sensitivity fluorescence polarization immunoassay method (y-int = 0.03 and 0.08, respectively). However, the modified EMIT assay had the best correlation in the low range (0.03-2 μmole/L). Additionally, endogenous and chemical interference testing demonstrated that the modified assay was not affected to a clinically significant extent. The described modifications have enhanced the sensitivity of the Syva EMIT assay for MTX measurements down to 0.05 μmole/L with acceptable precision that can be used in clinical practice for monitoring MTX therapy.

Cyclosporine Effects on Pediatric Kidney Recipients

(1), entitled “Cyclosporine trough and 2 hour post dose monitoring and its contributing factors among pediat-ric kidney recipients,” recently published in the Nephro-Urology Monthly. The authors correctly mentioned that there is a negative relationship between cyclosporine A levels (C0 and C2) and serum creatinine. Based on their data, the mean of C0 levels (118 ± 65 ng/mL) was in the therapeutic range, which is an acceptable level of cyclo-sporine after 6 months using the EMIT (Enzyme multi-plied immunoassay technique) assay method (2). Then lower serum creatinine levels that better reflects graft function are predictable. Although in this study, the mean of C2 levels (471 ± 148 ng/mL) was lower than thera-peutic levels (600-800 ng/mL), patients had good graft function. This result can be explained by previous simi-lar studies in Iran in which most patients at therapeutic C2 levels presented with cyclosporine nephrotoxicity or the recipients did not reach target levels, so they didn’t recommend routine checking of C2 levels (2, 3). This may be due to genetic differences in drug metabolism in dif-ferent races which can lead to different peak levels or dif-ferent peak times (for example 3 or 4 hours after taking) in Iranian patients.Another factor that may impact on graft function con-sists of the donor’s demographic data such as type (ca-daveric

A rapid, simple and sensitive liquid chromatography-tandem mass spectrometry method for routine clinical monitoring of tacrolimus with the Waters Masstrak-immmunosuppressant kit

Advances in mass spectrometry instruments have led to increased utilization of high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and it would be necessary to standardize blood quantification of immunosuppressant drugs. The aim of the study was to validate and assess the robustness of an LC-MS/MS method for quantification of tacrolimus in whole blood using the Waters Masstrak™ Immunosuppressant Kit. After protein precipitation from whole blood samples, chromatographic separation was performed in 2 min. Detection was performed with a Waters Tandem Quadrupole MS Quattro Premier XE, operated in multiple-reaction monitoring in positive electrospray ionization mode. This method was validated and compared to Enzyme Multiplied Immunoassay Technique (EMIT) method in accordance with actual guidelines. The limit of quantification was 1.0 ng/mL and the calibration curve was linear to 27.6 ng/mL. Between-day and within-day trueness and precision were < 15% at three concentrations spanning the linear range. The EMIT assay showed an average positive bias of 28.3% compared with the LC-MS/MS. Internal and external quality control were always accepted and demonstrated the robustness of this method. In conclusion, we validated a rapid, simple and robust quantification of tacrolimus in blood samples with the Waters Masstrak™ Immunosuppressant Kit.

Falsely Elevated Whole Blood Tacrolimus Concentrations due to Interference in an Affinity Column-Mediated Immunoassay Method on Xpand Dimension

The whole blood concentration of tacrolimus is required for therapeutic drug monitoring of this immunosuppressive drug. Abnormal tacrolimus levels affect its efficacy or toxicity, leading to changes in its dosage. Here, we report analytical interference in the affinity column-mediated immunoassay tacrolimus method on the Xpand autoanalyzer in a kidney transplant human immunodeficiency virus-infected patient. Tacrolimus concentrations obtained by affinity column-mediated immunoassay are 3- to 7-fold higher than measurements with the enzyme multiplied immunoassay technique assay. The cause of this interference remains unknown. However, it would be necessary to identify this type of interference to measure tacrolimus concentration with another method to avoid analytical error, which may lead to a poor clinical outcome.

Calcineurin Inhibitor Determination in Whole Blood With the RXL Dimension Analyzer: A Useful Tool for Immunosuppressive Drug Monitoring

Routine monitoring of cyclosporine and tacrolimus levels is necessary to minimize adverse side effects and to ensure effective immunosuppression. The RXL Dimension apparatus conceived for ACMIA technologies is proposed to determine C0 and C2 cyclosporine levels and also tacrolimus levels in whole blood without any dilution or pretreatment using specific calibrators and Flex reagent cartridges (reagent stability: 72 hours for Neoral C0 and C2; 48 hours for tacrolimus). The assay ranges were between 25 to 500 ng/mL for C0; 350 to 2000 ng/mL for C2; and 1.2 to 30 ng/mL for tacrolimus. Within-run and between-day imprecision were <10% for cyclosporine. The coefficient of linearity was r 2 = .998 for C0, C2, and tacrolimus. Moreover, for cyclosporine and tacrolimus assays, the time for the first result was 20 minutes. Cyclosporine (C0, n = 152; C2, n = 54) and tacrolimus (n = 70) ACMIA assays were compared with enzyme-multiplied immunoassay technique (EMIT) cyclosporine and tacrolimus assays (V-Twin, Siemens ex-Dade Behring Laboratories) among 276 transplant patients: 119 kidney, 67 liver, 28 heart, and 62 bone marrow transplantations. Values obtained with the ACMIA assay were highly correlated with the EMIT assay for CsA C0 levels (ACMIA = 1.04 EMIT - 9.32; r 2 = .97); CsA C2 levels (ACMIA = 1.15 EMIT - 53.7; r 2 = .94); and tacrolimus levels (ACMIA = 0.93 EMIT - 0.16; r 2 = .93). In conclusion, the RXL Dimension analyzer is a useful tool for routine monitoring with a single method for C0 and C2 cyclosporine and tacrolimus level determinations in whole blood without any dilution or preanalytic treatment.

Models for the prediction of mycophenolic acid area under the curve using a limited-sampling strategy and an enzyme multiplied immunoassay technique in chinese patients undergoing liver transplantation

Background: An enzyme multiplied immunoassay technique (EMIT) provides convenient and accurate measurements of mycophenolic acid (MPA) concentrations for determination of immunosuppression during treatment with mycophenolate mofetil (MMF). No abbreviated model for estimating the full 12-hour MPA AUC using an EMIT assay in liver transplant recipients has been described previously. Objective: This study was conducted to determine the best model for predicting the MPA AUC using the EMIT method and a limited-sampling strategy in Chinese patients undergoing liver transplantation. Methods: The study enrolled consecutive liver transplant patients who were receiving MMF 1 g BID along with tacrolimus. A complete MPA pharmacokinetic profile was obtained for each patient on a single day, 7 to 14 days after transplantation. The EMIT method was used to determine MPA concentrations before dosing and at 0.5, 1, 1.5, 2, 4, 6, 8, 10, and 12 hours after dosing on the sampling day. Multiple linear regression analysis was used to evaluate potential models for estimating the full 12-hour MPA AUC. The accuracy and robustness of the models were evaluated using bootstrap analysis. Prediction error and prediction bias were calculated. Agreement between the estimated MPA AUC 0-12 and the full 12-hour MPA AUC was investigated using Bland-Altman analysis. Results: The study enrolled 48 Chinese liver transplant recipients (45 male, 3 female) with a mean (SD) age of 50 (12) years, mean weight of 64 (12) kg, and mean height of 169 (6) cm. Twenty-four models that included blood sampling at 1 through 4 time points were developed ( r 2 = 0.015–0.950). Four models with the highest r 2 values were selected; the lack of significant differences from the original dataset on bootstrap analysis indicated acceptable accuracy and robustness. The best model for predicting the MPA AUC 0-12 employed concentrations at 1, 2, 4, and 8 hours; 40 of 48 (83.3%) MPA AUC 0-12 values estimated using this model were within 15% of the full 12-hour MPA AUC. This model had a minimal mean prediction error (mean [SD], 0.27% [1.79%]) and mean absolute prediction error (8.83% [1.24%]). On Bland-Altman analysis, this model also had the best agreement between the estimated MAP AUC 0-12 and the full 12-hour MPA AUC, with a mean error of 9.02 mg · h/L. Conclusions: In this small group of Chinese liver transplant patients receiving MMF and concomitant tacrolimus, models for estimating the MPA AUC 0-12 were developed using the EMIT method and a limited-sampling strategy. The best model for prediction of the full 12-hour MPA AUC was 4.46 + 0.81 · C1 + 1.78 · C 2+2.51·C 4+4.94·C8.

An Improved Application for the Enzyme Multipled Immunoassay Technique for Caffeine, Amikacin, and Methotrexate Assays on the Dade-Behring Dimension RxL Max Clinical Chemistry System

Caffeine is widely used in children's hospitals to treat neonatal apnea. Amikacin is used for treating hospital-acquired infections caused by Gram-negative bacteria resistant to other aminoglycosides. The blood levels, however, have to be monitored carefully because of its ototoxicity and nephrotoxicity. Methotrexate (MTX) is used as a chemotherapeutic agent in the treatment of leukemia and lymphoma as well as of certain solid tumors. Current enzyme multiplied immunoassay technique (EMIT) assays for caffeine, amikacin, and MTX lack low-end precision. In addition, the EMIT assays for MTX lack the sensitivity of reliable quantification to 0.05 micromol/L needed because of today's more rigorous requirements. The goal of the present study was to optimize the EMIT method parameters on the Dimension RxL Max, thereby providing applications with improved precision for all the three analytes and enhancing the sensitivity of the EMIT methotrexate assay. Serum samples were measured for caffeine, amikacin, and MTX by EMIT on the Dimension RxL Max and by EMIT (on the Olympus AU 600 for caffeine) and fluorescence polarization immunoassay [for MTX and amikacin (FPIA; TDx FLx)] at Quest Diagnostics. The new instrument method parameters that use larger sample volumes and longer observation of optical density changes (caffeine, MTX) provide improved sensitivity for MTX permitting reliable measurement at 0.05 micromol/L and improved precision for all three analytes. Within- and between-day imprecision were less than 6% for low to high concentrations of caffeine and amikacin controls and are less than 7.5% for MTX concentrations greater than 0.3 micromol/L and 12.3% at 0.06 micromol/L. The correlation coefficients for caffeine, amikacin, and MTX plotted for the Dimension RxL Max versus the methods used at Quest Diagnostics were 0.973, 0.986, and 0.992, respectively. These EMIT method applications now compare well with other established assays. The new Dimension RxL Max method parameters provide greatly improved precision and also meet today's clinical sensitivity guidelines (0.05 micromol/L) for MTX.

Comparison of Liquid Chromatography-Tandem Mass Spectrometry with a Commercial Enzyme-Multiplied Immunoassay for the Determination of Plasma MPA in Renal Transplant Recipients and Consequences for Therapeutic Drug Monitoring

Mycophenolic acid (MPA) is an immunosuppressive drug partly metabolized to MPA-glucuronide (MPAG), which is pharmacologically inactive. The currently available enzyme-multiplied immunoassay technique (EMIT) has been reported to overestimate MPA plasma concentration in clinical samples when compared with HPLC techniques. The aims of this study were to design and validate a specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique for the determination of MPA and MPAG using a low plasma volume and a simple sample preparation procedure; then to compare it with EMIT for the determination of MPA in plasma samples collected over an interdose interval at different posttransplantation periods (days 3, 7, and 30 and after 3 months) in 25 renal transplant recipients orally administered cyclosporine and mycophenolate mofetil twice daily, to investigate the origins of the differences between techniques. The LC-MS/MS technique developed showed limits of quantification (LOQs) of 0.1 mg/L and 1 mg/L for MPA and MPAG, respectively, and was linear, accurate, and precise from these LOQs up to 30 mg/L for MPA and 300 mg/L for MPAG. EMIT gave similar results to LC-MS/MS for spiked quality control samples (in a synthetic matrix or in drug-free plasma) but significantly overestimated MPA levels in clinical samples: EMIT - LC-MS/MS = +61.39% +/- 57.94%, with large variations depending on patients, time elapsed since transplantation, sampling time, and concentration levels. These results confirmed the known overestimation of the EMIT assay compared with a specific method and showed that the magnitude of this overestimation depended on sampling time and time after transplantation.

The EMIT 2000 tacrolimus assay: an application protocol for the Beckman Synchron LX20 PRO analyzer

Objective: To develop and evaluate the performance of an application protocol for the EMIT 2000 tacrolimus (Tac) assay on the Beckman Synchron LX20 PRO Analyzer. Design and Methods: Precision, accuracy, linearity, and lower limit of quantitation were investigated. Specimens from 212 kidney, liver, heart/heart–lung, and islet cell transplant patients were analyzed and results were compared to those from the Abbott MEIA II assay. A separate population of 232 specimens was coanalyzed by the enzyme-multiplied immunoassay technique (EMIT) assay and liquid chromatography tandem mass spectrometry (LC-MS/MS). Results: Total imprecision was 13.7% and 6.0% at concentrations of 3.4 and 19.1 μg/L, respectively. Recoveries from assayed reference materials ranged from 103% to 109%. A quantitation range of 3.2–30.0 μg/L was validated. The EMIT assay on the LX20 PRO analyzer showed an average negative bias of 1% compared to the MEIA assay and an average positive bias of 17% compared to LC-MS/MS. Conclusion: This application for the EMIT 2000 Tac assay on the Beckman Synchron LX20 PRO analyzer enhances the versatility of the immunoassay for routine therapeutic drug monitoring (TDM) of this immunosuppressant in the clinical setting.

Vancomycin assay performance in patients with acute renal failure.

Fluorescence polarization immunoassays (FPIA) have been reported to overestimate vancomycin serum concentrations compared to high-performance liquid chromatography (HPLC) or enzyme multiplied immunoassay technique (EMIT) in patients with chronic renal disease. The assay manufacturer has modified the FPIA to remedy this overestimation. The purpose of this study was to compare the assay performance of two FPIAs to EMIT in acute renal failure patients receiving vancomycin and continuous venovenous hemofiltration. Open-label trial. Intensive care unit in a university affiliated hospital. 15 serum and ultrafiltrate samples were obtained from 14 critically ill patients (mean +/- SD; 57 +/- 12 years; 8 males/6 females). Vancomycin concentrations were determined by a polyclonal FPIA (pFPIA) performed on the TDx system, a monoclonal FPIA (mFPIA) performed on the AxSYM system and EMIT. The coefficient of variation for all assays was < 5%. The mean difference +/- SDd between mFPIA vs EMIT and pFPIA vs EMIT assays in serum were: -0.08 +/- 1.55 and 1.24 +/- 2.11 mg/l, respectively. The limits of agreement between the mFPIA vs EMIT and pFPIA vs EMIT assays in serum were: -3.18 to 3.03 and -2.99 to 5.46 mg/l, respectively. Our data demonstrate that the manufacturer's changes to the pFPIA have reduced overestimation. The mFPIA appears to be an acceptable assay for measuring vancomycin serum concentrations in acute renal failure patients and does not significantly overestimate these concentrations.

Effect of Heating Sera under Conditions Necessary for Deactivation of Human Immunodeficiency Virus on Commonly Monitored Therapeutic Drugs

Minimizing the risk of infection of laboratory staff from contaminated blood samples is a major safety goal in a clinical laboratory. One dangerous pathogen, the human immunodeficiency virus (HIV) can be deactivated by heating sera at 56 degrees C for 30 min. We studied the effect of such heat treatment on serum concentrations of 11 commonly monitored therapeutic drugs. We used blood specimens collected in serum separator tubes (SSTs), which were routinely submitted for therapeutic drug monitoring in our laboratory for this study. Concentrations of digoxin in sera were measured using a fluorescence polarization immunoassay (FPIA), while concentrations of tobramycin, gentamicin, vancomycin, theophylline, valproic acid, procainamide, N-acetylprocainamide (NAPA), phenytoin, phenobarbital, and carbamazepine were measured by enzyme-multiplied immunoassay technique assays using a Monarch 2000 analyzer. We observed no significant change in the concentration of any drug except phenytoin and carbamazepine following heating at 60 degrees C. The decrease in concentration of phenytoin and carbamazepine after heating was related to absorption of the drug to the gel rather than the instability of the drug under heating conditions. We conclude that blood contaminated with HIV may be deactivated by heating prior to analysis for most of the routinely monitored therapeutic drugs.

Determination of Cocaine Usage in Pregnant Women by a Urinary EMIT Drug Screen and GC-MS Analyses

In mothers who had no prenatal care and in their newborns, the presence of cocaine and benzoylecgonine (BE) was determined in urine, hair, and meconium. Samples of urine and hair were obtained from pregnant women who entered the hospital for delivery. Cocaine usage was assessed by a urinary enzyme-multiplied immunoassay technique (EMIT) and by gas chromatography-mass spectrometry (GC-MS). GC-MS was used to detect the presence of cocaine and BE in maternal urine and hair and in meconium and hair obtained from their newborns. In this study of 40 women, the EMIT assay for urinary BE identified 17 (42.5%) of the women as having used cocaine. Of these 17 women, all of their newborns were exposed to cocaine during gestation, based on the analysis of neonatal hair and meconium for cocaine or BE. From the maternal samples that were assayed for cocaine and BE by GC-MS, it appears that hair analysis identified the most cocaine users (70%) of the 40 women who participated in the study. When GC-MS was used to analyze the various samples from mothers and their newborns, 80% of the neonates showed exposure to cocaine. This study shows that women with no prenatal care who have a positive urinary drug screen by EMIT for BE have exposed their newborns to cocaine. The data from pregnant women with a negative drug screen for BE show that 52.2% of their newborns had prior fetal exposure to cocaine.

Controlled release infusion kinetics of tobramycin.

Ten healthy male volunteers received two doses of tobramycin (2 mg/kg) in a crossover fashion, first by intravenous piggyback (IVPB), then by the CRIS infusion system after a washout period. Serum samples were drawn both during and after the infusions. Twenty-four-hour urine collections were assayed for tobramycin. Residual fluid from the lines of both delivery systems was measured and assayed for tobramycin concentration. All samples were run in duplicate, using an enzyme-multiplied immunoassay technique assay. The results indicate that there was a statistically higher amount of drug delivered via the CRIS system (98.3 +/- 0.3% versus 90.4 +/- 2.3%). No significant difference was found in urinary recovery between the two groups. Peak serum levels were significantly higher with the CRIS system, with 8/10 subjects having at least one serum level greater than 10 micrograms/ml, as compared to 0/10 when given by IVPB. Peak serum levels occurred at 30 min in all subjects given tobramycin through the CRIS system, compared to 50-60 min when delivered by IVPB. This difference in peak serum levels is primarily related to the rate of drug delivery and to the difference in the dose delivered to each subject. The significance of the serum concentration profiles is discussed.

Fluorescence polarization immunoassay evaluated for screening for amphetamine and methamphetamine in urine.

We studied the recently developed Abbott fluorescence polarization immunoassay (FPIA) for amphetamine and methamphetamine in urine and compared the results with those of the Syva enzyme-multiplied immunoassay technique (EMIT) and a gas-chromatographic assay. The FPIA method showed a limit of quantification of 0.3 mg/L, comparable with the lower cutoff of the EMIT assay. FPIA demonstrated greater specificity than the EMIT assay: phenylpropanolamine and ephedrine showed extremely limited cross reactivity with the FPIA antibody. Analysis of 249 urine specimens by all three methods clearly demonstrated the FPIA method to be acceptable for screening for amphetamine and methamphetamine in urine.