Cloned Enzyme Donor Immunoassay Research Articles

Update on Urine Adulterants and Synthetic Urine Samples to Subvert Urine Drug Testing.

To avoid a positive urine drug test, donors might try to subvert the test, either by adulterating the specimen with a product designed to interfere with testing or by substituting the specimen for a synthetic urine. A market search conducted in December of 2020 identified 3 adulterants and 32 synthetic urines, and a selection was procured based on specific criteria. Samples prepared with the 3 adulterants and 10 synthetic urines were submitted for testing at five forensic drug testing laboratories to perform immunoassay screening, chromatographic confirmation analysis and specimen validity testing (SVT). One adulterant determined to contain iodate reduced THC-COOH concentrations by 65% and the concentrations of 6-acetylmorphine, morphine, oxycodone, oxymorphone, hydrocodone and hydromorphone by 6-27%. Another adulterant determined to contain nitrite reduced THC-COOH concentrations by 22%, while the third did not affect drug screening or confirmatory testing. Both active adulterants could be identified through positive oxidant screens as well as through signal suppression in cloned enzyme donor immunoassay (CEDIA). The synthetic urines could not be identified either through traditional SVT or by the AdultaCheck10 dipstick. The Synthetic UrineCheck dipstick produced a difference in response between the authentic urine specimen and the synthetic urine samples, but the difference was small and difficult to observe. While most synthetic urines now contain uric acid, magnesium and caffeine, the results indicated that a biomarker panel including endogenous and exogenous markers of authentic urine performed well and clearly demonstrated the absence of biomarkers in the synthetic urines. The SVT assay also offers potential targets for future screening assays.

Microfluidic Device with an Integrated Freeze-Dried Cell-Free Protein Synthesis System for Small-Volume Biosensing.

Microfluidic devices enable the precise operation of liquid samples in small volumes. This motivates why microfluidic devices have been applied to point-of-care (PoC) liquid biopsy. Among PoC liquid biopsy studies, some report diagnostic reagents being freeze-dried in such microfluidic devices. This type of PoC microfluidic device has distinct advantages, such as simplicity of the procedures, compared with other PoC devices using liquid-type diagnostic reagents. Despite the attractive characteristic, only diagnostic reagents based on the cloned enzyme donor immunoassay (CEDIA) have been freeze-dried in the microfluidic device. However, development of the PoC device based on the CEDIA method is time-consuming and labor-intensive. Here, we employed a molecule-responsive protein synthesis system as the diagnostic reagent to be freeze-dried in the microfluidic device. Such molecule-responsive protein synthesis has been well investigated in the field of molecular biology. Therefore, using the accumulated information, PoC devices can be efficiently developed. Thus, we developed a microfluidic device with an integrated freeze-dried molecule-responsive protein synthesis system. Using the developed device, we detected two types of bio-functional molecules (i.e., bacterial quorum sensing molecules and mercury ions) by injecting 1 µL of sample solution containing these molecules. We showed that the developed device is applicable for small-volume biosensing.

Kavain Interference with Amphetamine Immunoassay.

We encountered unexpected false positive urine results in three patients for amphetamine-type substances by immunoassay, measured as part of community drug prevention programmes. Kavain was identified in all three urine samples by gas chromatography mass spectrometry (GC-MS). No other potential cross-reactants were found.Kavain is a kava-lactone present in kava, a ceremonial and recreational drink derived from roots and stem of the plant Piper Methysticum. It is consumed regularly by many indigenous Pacific and Australian Aboriginal communities. Urine immunoassay was performed on a Beckman Coulter AU480 Analyser using CEDIA amphetamine-type substances reagent and DRI ethanol reagent. Three different kava powders were purchased from local kava clubs and dissolved in ethanol, then evaporated and reconstituted in blank urine and analysed by immunoassay, GC-MS for amphetamine-type substances. Additionally authentic kavain standard was also tested for cross reactivity by immunoassay and analysed by GC-MS to compare the mass fragmentation pattern and retention time with the kava powder and patient specimens. The patient urine samples tested positive by CEDIA immunoassay for amphetamines. However, when analysed by GCMS they were negative for amphetamine-type but contained kavain.The kava powders and kavain standard all cross reacted with the amphetamine immunoassay to give falsely detected results. GCMS did not identify any amphetamine-type compounds in any of the Kava powders nor in the kavain standard. To our knowledge, this is the first report of false positive amphetamine measurements due to kavain, a component of the kava drink, widely consumed in Oceania and Australasia.

Analytical Detection of Novel Stimulants by Immunoassay and Liquid Chromatography-High Resolution Mass Spectrometry: Case Studies on Ethylphenidate and Mephedrone.

The advent of hundreds of new compounds aimed at the substance misuse market has posed new analytical challenges. A semi-quantitative liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method has been developed to detect exposure to two novel stimulants, mephedrone and ethylphenidate, and selected metabolites. Centrifuged urine (50 µL) was diluted with LC eluent containing internal standards (mephedrone-d3, methylphenidate-d9 and ritalinic acid-d10; all 0.02 mg/L) (450 µL). Intra- and inter-assay accuracy and precision were within ±15% and <6%, respectively, for all analytes. The limit of detection was 0.01 mg/L for all analytes. Urine samples from mephedrone and ethylphenidate users were analyzed using immunoassay (amphetamine-group cloned enzyme donor immunoassay [CEDIA]) and LC-HRMS. Ethylphenidate, mephedrone and selected metabolites all had low cross-reactivity (<1%) with the immunoassay. The median (range) amphetamine-group CEDIA concentration in urine samples from mephedrone users (n = 11) was 0.30 (<0.041-3.04) mg/L, with only 1 sample giving a positive CEDIA result. The amphetamine-group CEDIA concentration in the urine sample from an ethylphenidate user was <0.041 mg/L. Improving the detection of novel compounds is of increasing importance to enable accurate diagnosis and treatment. Immunoassay methods used for drug screening may be inappropriate and lead to false-negative results. Conversely, detection of these compounds is possible through use of LC-HRMS and can provide information on the metabolites present after exposure to these drugs.

Cross-reactivity of nefopam and its metabolites on three different urine benzodiazepines immunoassay screenings: A comparative study

Summary Objectives To highlight an analytical interference involving nefopam and its metabolites on urinary benzodiazepine screening with Enzyme Multiplied Immunoassay Technique (EMIT) Atellica CH from Siemens Healthineers, and to evaluate two other immunoassays: MULTIGENT on Architect from Abbott Diagnostics and Cloned Enzyme Donor Immunoassay (CEDIA) Thermo Fisher kits on Cobas from Roche Diagnostics. Case/methods An analytical interference was suspected for a 58-year-old-woman hospitalized for unexplained malaise. The urinary EMIT Atellica benzodiazepine immunoassay was positive. Screenings by liquid chromatography coupled with diode array detector (LC-DAD) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) identified nefopam and metabolites but no benzodiazepine. Drug-free urine has been spiked at different concentrations of nefopam (187,5–3000 μg/L) and tested on EMIT Atellica assay. Three urines from nefopam-treated patients and two negative controls have been selected to assess this cross-reactivity. Nefopam quantification has been performed by LC-DAD. Different immunoassays were evaluated: EMIT on Atellica, MULTIGENT on Architect and CEDIA on Cobas. Results Drug-free urines spiked with nefopam were negative on EMIT Atellica immunoassay. The interference observed was confirmed for nefopam-treated patients (2/3) on EMIT Architect. For one patient, an interference was observed with a low concentration of nefopam ( Discussion/conclusion Comparison of three immunoassay techniques revealed a heterogeneity of the interference. Nefopam metabolites may be responsible for this cross-reactivity, suggesting the importance of testing them during the kits evaluation phase. This study highlights the need to confirm immunoassay results by more specific techniques.

Optimization of cloned enzyme donor immunoassay cut-offs for drugs of abuse in post-mortem whole blood

IntroductionImmunoassay (IA) tests are not widely applied in post-mortem samples, since they are based on technologies requiring relatively non-viscous specimens, and compounds originating from the degradation of proteins and lipids during the post-mortem interval can alter the efficiency of the test. However, since the extraction techniques for IA tests are normally rapid and low-cost, IA could be used as near-body drug-screening for the classes of drugs most commonly found in Italy and Europe. In this study, semi-quantitative results on post-mortem whole blood samples obtained through CEDIA analysis (cannabinoids, cocaine, amphetamine compounds, opiates and methadone), were compared with results of confirmatory analysis obtained using GC–MS. Screening cut-offs for all drugs were retrospectively optimized. MethodsPost-mortem whole blood samples from autopsy cases of suspected fatal intoxication were collected over 3 years. Samples were initially analyzed through CEDIA (CEDIA, ILab 650, Werfen). Confirmatory analyses were then performed by GC–MS (QP 2010 Plus, Shimadzu). Screening cut-offs were retrospectively optimized using Receiver Operating Characteristic (ROC) analysis. ResultsCEDIA results were available for 125 samples. Two-hundred-eighty-nine (289) positive screening results were found. Among these, 162 positive confirmation results were obtained. Optimized screening cut-offs were as follows: 6.5ng/ml for THC; 4.2ng/ml for THC-COOH; 12.0ng/ml for cocaine; 6.6ng/ml for benzoylecgonine; 6.4ng/ml for opiates; 2.0ng/ml for methadone. Analysis of ROC-curves showed a satisfying degree of separation in all tests except for amphetamine compounds, with areas under the curve (AUC) between 0.915 (THC) and 0.999 (for benzoylecgonine and methadone). DiscussionThe results of the study showed that CEDIA screening at the optimized cut-offs exhibits a very high sensitivity and good specificity and positive predictive value (PPV) for cannabinoids, cocaine and metabolites, opiates and methadone. A high number of false positives (n=19) for amphetamine compounds was observed at the optimized cut-off, resulting in a very low PPV, which is also influenced by the very low number of TP (n=4). ConclusionThe results of the study show that the CEDIA is a valuable screening test on post-mortem whole blood for cannabinoids, cocaine and metabolites, opiates and methadone, but it is not recommended for amphetamine compounds, due to the high number of false positives. The strengths of the study are the large sample size, the inclusion of post-mortem cases only and the high level of sensitivity and specificity obtained at the optimized cut-offs.

Development of a microdevice for facile analysis of theophylline in whole blood by a cloned enzyme donor immunoassay.

We have developed a microdevice for therapeutic drug monitoring. In this device, dispensing of sample and reagent was accomplished by simple manual operation of a syringe. Moreover, for a simple and rapid measurement, we used cloned enzyme donor immunoassay as a detection principle. These features and the reagent that is enclosed in microdevice beforehand make it possible to complete the facile analysis. In this paper, our model analyte was 1,3-dimethylxanthine (theophylline), a kind of bronchodilator. The fluorescence measurement of theophylline in whole blood was achieved with the limit of detection of 0.73 μg mL-1. This microdevice provides rapid analysis (4 min), requires only a small volume of sample (2 μL) and features simple operation; hence, it is readily applicable to point of care testing.

Optimization of cloned enzyme donor immunoassay cut-offs for drugs of abuse in whole blood of drivers involved in road accidents

IntroductionImmunoassay (IA) tests should be able to detect low concentrations of illegal drugs when used for the screening of drugs in drivers. False negatives should be avoided, and false positives should be reduced as far as possible. In this study, semi-quantitative results for blood samples containing illicit drugs (cannabinoids, cocaine, amphetamines/methamphetamines, opiates and methadone) obtained with cloned enzyme donor immunoassay (CEDIA), were compared with results of confirmatory analysis performed through gas chromatography-mass spectrometry (GC–MS). Screening cut-off points for each class of drugs were retrospectively optimized. MethodsWhole blood samples from drivers involved in road accidents in the period from January 2013-December 2017 were analyzed with CEDIA (4200 samples). Confirmatory analyses were performed through (GC–MS) on: (i) all samples with screening concentrations above 1ng/ml for at least one drug (positive screening results); (ii) 800 samples with screening concentration lower than 1ng/ml (negative screening results). Recommended per se limits in relation to driving under the influence of drugs were set as fixed values. Sensitivity, specificity, positive and negative predictive values were evaluated by contingency tables and compared to ROC-analysis in order to obtain ideal screening cut-offs. ResultsCEDIA results were available for 4200 blood samples and 1172 positive screening results were found. Among these, 1008 confirmation analysis were obtained through GC–MS. Optimized screening cut-offs obtained through ROC analysis were as follows: 8.0ng/ml for THC; 5.5ng/ml for THC-COOH; 21.1ng/ml for cocaine; 6.9ng/ml for benzoylecgonine; 33.1ng/ml for opiates; 61.6ng/ml for amphetamines; 5.0ng/ml for methadone. Using these cut-offs, sensitivity was above 97% for THC-COOH, cocaine, benzoylecgonine, amphetamines, opiates and methadone, and 92% for THC; specificity was above 90% for cocaine, benzoylecgonine, amphetamines, opiates and methadone, 80% for THC and 89% for THC-COOH; negative predictive value was above 99% for all drugs and metabolites. ConclusionPrevious studies have shown that CEDIA tests are useful for preliminary screening of serum and urine. Its implementation in whole blood is of primary importance for the assessment of impaired driving, since the per se limits of many European countries refer to whole blood, and preparation of the serum and/or the collection of urine is not always possible in the hospital emergency department, where blood samples are withdrawn. Our study shows that CEDIA tests on whole blood permit the definition of cut-off values with optimal sensitivity and negative predictive values for all analytes (near to 100%), including very good specificity.

Bufotoxin poisoning that showed the sign of acute digitalis overdose in the patient of Kyushin® intoxication

Introduction: Kyushin® is a widely used herbal medicine in East Asia for heart failure. Toad venom in present in Kyushin®, which has positive inotropic effect as digitoxin. Case presentation: An 81-year-old, female patient presented with decreased mental status after overdose of Kyushin®. The first electrocardiogram showed junctional tachycardia with 142/min, suddenly dropped to 27/min and followed by ventricular fibrillation. After one cycle of CPR, spontaneous circulation returned but junctional bradycardia, tachycardia, and ventricular fibrillation appeared. After six times of defibrillation, spontaneous circulation returned, and mechanical ventilator and transcutaneous pacing were applied. Plasma toxicology test revealed digitoxin 66.90 ng/mL by cloned enzyme donor immunoassay and digoxin 0.76 ng/mL by kinetic interaction of microparticles in solution immunoassay. After 8 h from presentation, the patient’s mental status came to be alert, and then transcutaneous pacing was removed. Discussion: Cloned enzyme donor immunoassay has been reported to be highly cross-reactive with digoxin-like substances, which strongly supports bufotoxin to be responsible for arrhythmia of the patient. Conclusion: Poisoning of bufotoxin in Kyushin® can cause cardiac arrhythmia, even arrest, but without digoxin-specific Fab, conventional therapy could be successful.

Influence of uridine diphosphate-glucuronosyltransferases (1A9) polymorphisms on mycophenolic acid pharmacokinetics in patients with renal transplant

Background: There are differences in pharmacokinetic of mycophenolic acid among individuals. The UGT1A9 enzyme is of special interest since it is the main enzyme involved in the glucuronidation of MPA. Single nucleotide polymorphisms in the UGT1A9 gene may be responsible for individual differences in the pharmacokinetics of MPA. The aim of this study was to explain MPA pharmacokinetics in UGT1A9 1399 C > T polymorphisms in Turkish renal transplant patients. Patients and methods: One hundred and twenty-five living-donor transplant recipients and 100 healthy control subjects underwent UGT1A9 1399 C > T genotyping using polymerase chain reaction–restriction fragment length polymorphism. Concentrations of MPA were determined with Cloned Enzyme Donor Immunoassay (CEDIA). Besides that, all the patients were monitored for acute rejection and graft function during the study period. Results: The UGT1A9 1399 C > T CC, CT, and TT genotype frequencies among patients were, respectively, 68.0%, 23.2%, and 8.8%. The CC, CT, and TT genotype frequencies among controls were, respectively, 63.0%, 23.0%, and 14.0%. There was no significant difference between patients and controls (p = .480, p = .999, p = .286, respectively). At first month, respectively, through blood concentrations of MPA were significantly higher in UGT1A9 1399 C > T TT carriers than in CT and CC carriers (p = .046). The doses for these patients were lower at first month (p = .021). Acute rejection episodes were not associated with the CC vs CT or TT genotypes (p = .064). Conclusions: Our results demonstrated a correlation between the UGT1A9 1399 C > T polymorphism and MPA pharmacokinetics among renal transplant patients. Determination of UGT1A9 polymorphism may help to achieve target of MPA blood concentrations.

Regional drug abuse prevalence depending on laboratory based urine illicit drug screening results

Objectives: To determine the laboratory based drug abuse prevalence of persons who have illicit and/or addictive substance test requests instead of alcohol in a city in Middle Anatolia in Turkey. Methods: Laboratory information system’s data of urine samples, submitted to Biochemistry Laboratory from various clinics, between January 2014 and June 2016 were retrospectively investigated. In comprehension of illicit drug use amphetamin, cannabis, opiate, cocaine and benzodiazepin group tests were screened with Cloned Enzyme Donor Immunoassay method. Positivity-negativity rates according to years, age-gender distribution and number of multiple substance usage were investigated. Results: Totally 79873 illicit drugs were screened in 19763 urine samples. 18080 males (% 91.5) and 1683 females (%8.5), with a mean age of 30.7±10.8 years (range: 4-86) and 27.2±10.5 years (range: 1-86) respectively were included. The average positivity rate according to the sample size in last 2.5 years was 23%; 19% in 2014, 23% in 2015, and 27% in 2016. The positivity rates according to number of analyzed tests were 5.9%, 6.7% and 7.5% respectively. In 2014 the maximum positivity rate was in benzodiazepine (12.5%) and secondly in cannabinoids (11.9%). Whereas amphetamine were the most common drug used (16.3%) with a 4.17 fold increase in 2015 compared to 2014. The increase in amphetamine group substance positivity continued in the first half of 2016 compared to previous year. The most common and the second most common multiple substance usage were amphetamine+ cannabis and amphetamine+opiate respectively. Conclusion: According to laboratory information of illicit drug screening results in urine, drug abuse is increasing through years. Amphetamine group is the most common used substance in the last two years in this region. This is the first study contributing to the prevalence of substance use in a city of Middle Anatolia in Turkey with a large sample volume and patient population.

Influence of Proton Pump Inhibitors on Mycophenolic Acid Pharmacokinetics in Patients With Renal Transplantation and the Relationship With Cytochrome 2C19 Gene Polymorphism

BackgroundMost patients have serious digestive complications after renal transplantation. Therefore, it is important to protect gastrointestinal function to improve the survival rate of transplant patients. Proton pump inhibitors (PPIs) such as lansoprazole and rabeprazole are widely administered to renal transplant patients with mycophenolic acid (MPA) in the perioperative period. PPIs are metabolized by cytochrome (CYP) 2C19 enzymes. Mycophenolate sodium (MYF) and mycophenolate mofetil (MMF) have been used in immunosuppression. Clinically relevant drug-drug interactions have been described between immunosuppressive drugs. In the present study, we investigated the drug interaction between MPA and lansoparazole or rabeprazole and the impact of CYP2C19 polymorphisms on these drug interactions after renal transplantation. Materials and MethodsA total of 125 renal transplant patients taking MPA derivatives between 2012 and 2016 were included in this study. The 125 patients were divided into 6 groups: MMF/tacrolimus/steroid together with lansoprazole or rabeprazole; MYF/tacrolimus/steroid together with lansoprazole or rabeprazole and without PPI. The single nucleotide polymorphisms of CYP2C19 were determined by the polymerase chain reaction-restriction fragment length polymorphism. Plasma concentrations of MPA were measured by cloned enzyme donor immunoassay. Clinical parameters such as incidence of delayed graft function and acute rejection, the rate of change of serum creatinine, toxicity, and gastrointestinal adverse effects were analyzed retrospectively. ResultsThe mean concentrations of MPA in the MYF group were higher than those in the MMF group. The mean dose-adjusted blood concentration of MPA coadministered with lansoprazole was lower than that of MPA with rabeprazole or without PPI in MMF and MYF groups (P < .05). In patients with the CYP2C19*2/*2 genotype, the mean concentrations of MMF with lansoprazole were significantly lower than those with rabeprazole with MMF or without PPI (P < .05). Gastrointestinal side effects were significantly higher in MMF with lansoprazole group than in MYF with lansoprazole group (P < .05). However, no differences were found according to genotype distribution in all groups (P > .05). ConclusionPolymorphisms in CYP2C19 are related to the metabolic oxidation of drugs to varying degrees. Both genetic and clinical factors in pharmacokinetics may help to make further progress toward individualized therapy to yield maximum efficacy with minimal side effects.

Illicit substance use among persons admitted to probation polyclinic of a regional mental hospital in the Eastern Anatolia, Turkey.

Objectives:To investigate the illicit substance use trends by gender and year in Eastern Turkey, Elazığ.Methods:This is a retrospective study designed to assess the drug use prevalence in persons who admitted to the Probation Policlinic of Elazig Mental Health Hospital between January 2011 and December 2014 in Eastern Turkey, Elazığ. Laboratory screening tests for drugs were studied using the cloned enzyme donor immunoassay technique in urine.Results:The study consisted of 10267 males (95.3%) and 510 females (4.7%), with a mean age of 30.42±10.83 years. Overall prevalence was 32.1% for cannabis, 3.8% for opiate, 0.35% for cocaine and 1.2% for polydrug usage. The prevalence of cannabis was significantly higher in males (32.7%, p=0.000). The prevalence of cannabis use was the lowest in the age group of 50-59 while it was significantly higher in the age groups of 20-29 and 30-39 (p=0.000). The prevalence of opiate use showed a significant difference among the years (p=0.000). There was a significant difference in the prevalence of cocaine use among the years (p=0.02). The prevalence of polysubstance use showed a significant difference among years (p=0.000).Conclusion:Cannabis was the most common illicit drug in the east of the Turkey. Further studies are required for comparing the results of various regions of the country and developing early interventions and treatment facilities.

Cross-reactivity of the CEDIA buprenorphine assay in drugs-of-abuse screening: influence of dose and metabolites of opioids.

PurposeThe cloned enzyme donor immunoassay (CEDIA) for buprenorphine is applied for both urine drugs-of-abuse screening and compliance monitoring. Sensitivity, specificity, and optimal cutoff of this assay have differed between studies. This may indicate that cross-reactivity has to be taken into account during assay evaluation. We therefore investigated the performance of the CEDIA buprenorphine assay for use in our patient population and explored the impact of cross-reactivity on assay accuracy.MethodsThe CEDIA buprenorphine assay and high-performance liquid chromatography–tandem mass spectrometry were employed to analyze drugs-of-abuse in urine samples from a healthy drug-naïve male volunteer after intake of two tablets of a prescription drug containing 400 mg paracetamol +30 mg codeine phosphate, and in urine samples (n=2,272) from drug-addicted patients. Receiver operating characteristic analyses were performed to express the diagnostic accuracy of the CEDIA buprenorphine assay.ResultsCEDIA buprenorphine was positive in one urine sample from the drug-naïve person after intake of the prescription drug. Twenty-five (1.1%) of the patient urine samples were positive for buprenorphine by CEDIA, but negative by high-performance liquid chromatography–tandem mass spectrometry. Codeine, morphine, and their respective metabolites were prevalent in samples that were false positive for buprenorphine. The specificity of the CEDIA buprenorphine assay increased to 99.7% when the cutoff was increased from 5 ng/mL to 10 ng/mL.ConclusionIntake of a therapeutic dose of codeine can yield a false-positive CEDIA buprenorphine result. Additive effects from metabolites of codeine contribute to cross-reactivity in concentrations much lower than listed in the manufacturer’s cross-reactivity guide. Raising the cutoff from 5 ng/mL to 10 ng/mL increased the diagnostic accuracy. Clinicians should be informed about the risk of false-positive results with the CEDIA buprenorphine assay.

Genetic association of interleukin-2, interleukin-4, interleukin-6, transforming growth factor-β, tumour necrosis factor-α and blood concentrations of calcineurin inhibitors in Turkish renal transplant patients.

Cytokines are essential for the control of the immune response as most of the immunosuppressive drugs target cytokine production or their action. The calcineurin inhibitors (CNIs) cyclosporine (CsA) and tacrolimus are immunosuppressive drugs widely used after renal transplantation to prevent allograft rejection. They are characterized by large interindividual variability in their pharmacokinetics; therefore, monitoring their blood concentrations is important to predict their optimal dosage following transplantation. Calcineurin inhibitors inhibit the phosphatase activity of calcineurin, thereby suppressing the production of other cytokines such as transforming growth factor (TGF-β), tumour necrosis factor-α (TNF-α), interleukin (IL)-6, IL-2, and IL-4. The aim of this study was to investigate the relationship between polymorphisms of cytokines and blood concentrations of CNIs in renal transplant patients. The study included 53 CsA-treated renal transplant patients and 37 tacrolimus-treated renal transplant patients. Cytokine polymorphisms were analysed using polymerase chain reaction (PCR) sequence-specific primers with the cytokine CTS-PCR-sequence-specific primers Tray Kit; University of Heidelberg. Blood concentrations of CNIs were determined with Cloned Enzyme Donor Immunoassay (CEDIA) method. Patients with TC genotype of TGF-β at codon 10 had lower CsA blood concentrations than the TT and CC genotypes (P = 0.005) at 1 month in CsA treatment group. The ratio of blood concentration/dose of CsA for patients with TGF-β1-codon 10 TC genotype was lower than for patients with TT, CC genotypes, and the dose given to these patients was higher in the first month (P = 0.046). The ratio of blood concentration/dose of CsA for patients with IL-2-330 GG genotype was higher than for patients with GT, TT genotypes, and the dose given to these patients was lower at first month and sixth months (P = 0.043, P = 0.035 respectively). The tacrolimus blood concentrations were significantly higher in patients with the genotype GG of IL-2-330 (P = 0.012) at the third month. Patients who had the TC genotype TGF-β codon 10 had lower CsA blood concentrations and this group had higher acute rejection (P = 0.033). These results suggest that the genotyping for TGF-β-codon 10, IL-2-330 and IL-6-174 polymorphisms may help individualized immunosuppressive dosage regiments.

KIMS, CEDIA, and HS-CEDIA Immunoassays Are Inadequately Sensitive for Detection of Benzodiazepines in Urine from Patients Treated for Chronic Pain

Background: Patients treated for chronic pain may frequently undergo urine drug testing to monitor medication compliance and detect undisclosed prescribed or illicit drug use. Due to the increasing use and abuse of benzodiazepines, this class of medications is often included in drug screening panels. However, immunoassay-based methods lack the requisite sensitivity for detecting benzodiazepine use in this population primarily due to their poor cross-reactivity with several major urinary benzodiazepine metabolites. A High Sensitivity Cloned Enzyme Donor Immunoassay (HS-CEDIA), in which betaglucuronidase is added to the reagent, has been shown to perform better than traditional assays, but its performance in patients treated for chronic pain is not well characterized. Objectives: To determine the diagnostic accuracy of HS-CEDIA, as compared to the Cloned Enzyme Donor Immunoassay (CEDIA) and Kinetic Interaction of Microparticles in Solution (KIMS) screening immunoassays and liquid chromatography-tandem mass spectrometry (LC-MS/MS), for monitoring benzodiazepine use in patients treated for chronic pain. Study Design: A study of the diagnostic accuracy of urine benzodiazepine immunoassays. Setting: The study was conducted at an academic tertiary care hospital with a clinical laboratory that performs urine drug testing for monitoring medication compliance in pain management. Methods: A total of 299 urine specimens from patients treated for chronic pain were screened for the presence of benzodiazepines using the HS-CEDIA, CEDIA, and KIMS assays. The sensitivity and specificity of the screening assays were determined using the LC-MS/MS results as the reference method. Results: Of the 299 urine specimens tested, 141 (47%) confirmed positive for one or more of the benzodiazepines/metabolites by LC-MS/MS. All 3 screens were 100% specific with no false-positive results. The CEDIA and KIMS sensitivities were 55% (78/141) and 47% (66/141), respectively. Despite the relatively higher sensitivity of the HS-CEDIA screening assay (78%; 110/141), primarily due to increased detection of lorazepam, it still missed 22% (31/141) of benzodiazepine-positive urines. The KIMS, CEDIA, and HS-CEDIA assays yielded accuracies of 75%, 79%, and 90%, respectively, in comparison with LC-MS/MS. Limitations: This study was limited by its single-site location and the modest size of the urine samples utilized. Conclusions: While the HS-CEDIA provides higher sensitivity than the KIMS and CEDIA assays, it still missed an unacceptably high percentage of benzodiazepine-positive samples from patients treated for chronic pain. LC-MS/MS quantification with enzymatic sample pretreatment offers superior sensitivity and specificity for monitoring benzodiazepines in patients treated for chronic pain. Key words: High sensitivty immunoassay, benzodiazepine, beta-glucoronidase, pain management, compliance, LC-MS/MS, screening

Improved buprenorphine immunoassay performance after urine treatment with β-glucuronidase.

Buprenorphine (BUP), a semi-synthetic opioid analgesic, is increasingly prescribed for the treatment of chronic pain and opioid dependence. Urine immunoassay screening methods are available for monitoring BUP compliance and misuse; however, these screens may have poor sensitivity or specificity. We evaluated whether the pretreatment of urine with β-glucuronidase (BG) improves the sensitivity and overall accuracy of three BUP enzyme immunoassays when compared with liquid chromatography-tandem mass spectrometry (LC-MS-MS). Urine samples sent to our laboratories for BUP testing (n = 114) were analyzed before and after BG pretreatment by cloned enzyme donor immunoassay (CEDIA), enzyme immunoassay (EIA) and homogenous EIA (HEIA) immunoassays using a common 5 ng/mL cutoff. Total BUP and norbuprenorphine (NBUP) concentrations were measured by LC-MS-MS as the reference method. Urine BG pretreatment improved EIA, HEIA and CEDIA sensitivities from 70, 82 and 94%, respectively, to 97% for each of the three methods, when compared with LC-MS-MS. While the specificity of the EIA and HEIA remained 100% after BG pretreatment, the specificity of the CEDIA decreased from 74 to 67%. Urine pretreatment with BG is recommended to improve sensitivity of the EIA and HEIA BUP screening methods.

Comparison of Immunoassay Screening Tests and LC-MS-MS for Urine Detection of Benzodiazepines and Their Metabolites: Results of a National Proficiency Test

For most diverse purposes, different immunoassay (IA) screening methods are usually used to detect benzodiazepines and their metabolites in urine. In this study, we compared the main IAs used in forensic toxicology (Cloned Enzyme Donor Immunoassay, CEDIA®; Enzyme-Multiplied Immunoassay Technique, EMIT®; Fluorescent Polarization ImmunoAssay, FPIA®; Kinetic Interaction of Microparticles in Solution, KIMS® and Immunochromatographic Techniques, IMC) with liquid chromatography-tandem mass spectrometry (LC-MS-MS). Twelve urine specimens were analyzed by 178 laboratories in Italy that participated in a National Proficiency Test, providing both qualitative and semi-quantitative results. Each IA was evaluated by the parameters: true positive, true negative, false positive (FP), false negative (FN), sensitivity (SENS), specificity (SPEC), positive predictive value, negative predictive value (NPV) and accuracy. SPEC was affected by a high FP rate for all IAs. The lowest SENS and NPV were provided by FPIA due to a high number of FN cases. Comparing IA semi-quantitative data with LC-MS-MS results, an overestimation of benzodiazepine amount is noted. This paper draws attention to the problem of the careless use of IA tests for forensic purposes as they may provide FP and/or FN results that can lead to errors of great severity.

Mathematical Equations to Calculate True Mycophenolic Acid Concentration in Human Plasma by Using Two Immunoassays With Different Cross‐Reactivities With Acyl Glucuronide Metabolite: Comparison of Calculated Values With Values Obtained by Using an HPLC‐UV Method

Both immunoassays and chromatographic methods are available for therapeutic drug monitoring of mycophenolic acid (MPA). Although chromatographic methods are more precise, immunoassays are widely used in clinical laboratories due to ease of adopting such assays on automated analyzers. We studied the possibility of using mathematical equations to calculate true MPA concentration by accounting for acyl glucuronide cross-reactivities with immunoassays by using two immunoassays with widely different cross-reactivities with the metabolite. We determined MPA concentrations in 20 specimens obtained from transplant recipients using cloned enzyme donor immunoassay (CEDIA) assay and a new particle enhanced turbidimetric inhibition immunoassay (PETINIA) assay. Then we developed mathematical equations to calculate true MPA concentration using values obtained by both immunoassays and reported cross-reactivity of acyl glucuronide with respective immunoassays. Calculated concentrations were compared with values obtained by using a high-performance liquid chromatography combined with ultraviolet detection (HPLC-UV) method. We obtained good correlation between calculated MPA concentrations and corresponding MPA level obtained by using HPLC-UV method. Using x-axis as the MPA concentrations determined by the HPLC-UV method and y-axis as the calculated MPA level, we observed the following regression equation: y = 1.083x - 0.0995 (r = 0.99, n = 20). Mathematical equations can be used to calculate true MPA concentrations using two immunoassays with different cross-reactivities with acyl glucuronide metabolite.

Comparison of CEDIA FK506 Assay With HPLC/MS/MS in a Large Cohort of Pediatric Patients

FK506 (tacrolimus), a macrolide immunosuppressant, is widely used in pediatric transplant patients, but a relatively narrow therapeutic window in children vs adults requires close and accurate monitoring of whole blood FK506 levels. High-pressure liquid chromatography/tandem mass spectrometry (HPLC/MS/MS)-based assays have been viewed as the gold standard but are more time and labor intensive than cloned enzyme donor immunoassay (CEDIA). To analyze differences between the 2 assays, we assayed FK506 in 348 split samples simultaneously by both methods. A further 70 samples were stratified by organ transplantation type: cardiac (13%), renal (23%), small bowel (22%), or liver transplantation (42%). Results were analyzed using standard statistical techniques for method comparison. CEDIA overestimated the FK506 value relative to HPLC/MS/MS by more than 20% in 40% of cases (139/348), whereas CEDIA underestimated the FK506 value relative to HPLC/MS/MS by more than 20% in 13.5% of cases, for a total inaccuracy of 53% using a ±20% cutoff. Only 28% of samples (99/348) measured by CEDIA were within 10% of the value obtained by HPLC/MS/MS. Bland-Altman analysis showed a mean bias of 9.5% in favor of CEDIA over HPLC/MS/MS (95% confidence interval, 6.1%-12.9%). Positive bias was greatest for liver transplant and R(2) values were lowest for intestinal transplant patients, indicating that HPLC/MS/MS may be a better option for this pediatric transplant subgroup.