Development of a rapid and quantitative lateral flow assay for the simultaneous measurement of serum κ and λ immunoglobulin free light chains (FLC): inception of a new near-patient FLC screening tool.

BACKGROUND: Serum free light chains (FLC) are sensitive biomarkers used for the diagnosis and management of plasma cell dyscrasias, such as multiple myeloma (MM), and are central to clinical screening algorithms and therapy response criteria. We have developed a portable, near-patient, lateral-flow test (Seralite®) that quantitates serum FLC in 10 min, and is designed to eliminate sample processing delays and accelerate decision-making in the clinic. METHODS: Assay interference, imprecision, lot-to-lot variability, linearity, and the utility of a competitive-inhibition design for the elimination of antigen-excess ('hook effect') were assessed. Reference ranges were calculated from 91 healthy donor sera. Preliminary clinical validation was conducted by retrospective analysis of sera from 329 patients. Quantitative and diagnostic results were compared to Freelite®. RESULTS: Seralite® gave a broad competitive-inhibition calibration curve from below 2.5 mg/L to above 200 mg/L, provided good assay linearity (between 1.6 and 208.7 mg/L for κ FLC and between 3.5 and 249.7 mg/L for λ FLC) and sensitivity (1.4 mg/L for κ FLC and 1.7 mg/L for λ FLC), and eliminated anomalous results from antigen-excess. Seralite® gave good diagnostic concordance with Freelite® (Roche Hitachi Cobas C501) identifying an abnormal FLC ratio and FLC difference in 209 patients with newly diagnosed MM and differentiating these patients from normal healthy donors with polyclonal FLC. CONCLUSIONS: Seralite® sensitively quantitates FLC and rapidly identifies clinical conditions where FLC are abnormal, including MM.

Validation of the Cozart microplate EIA for analysis of opiates in oral fluid.

The purpose of this study was to determine the performance characteristics of the Cozart microplate EIA for detecting opiates in oral fluid from patients in a drug misuse treatment program. Oral fluid samples were collected using the Cozart RapiScan Collection System from 216 donors who were receiving treatment for their addiction and were monitored for drug abuse. A further 40 oral fluid samples were collected from volunteer donors who were not drug users. The samples were analyzed in the laboratory by using the Cozart microplate EIA for opiates and confirmed using gas chromatography-mass spectrometry (GC-MS). The samples were stored frozen until analysis by GC-MS. The intra-assay precision for the Cozart microplate oral fluid EIA for opiates over 40 assays was 0.43% to 9.13% CV (within assay) and 2.9% to 9.1% CV (within day). A total of 109 samples were positive for various opiates. The Cozart microplate EIA for opiates in oral fluid, using a cut-off of 30 ng/ml morphine equivalents in neat oral fluid, had a sensitivity of 99.1 +/- 2.1% and a specificity of 94.4 +/- 2.2% versus GC-MS. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.

Comparison of Cozart microplate ELISA and GC-MS detection of methadone and metabolites in human hair.

The purpose of this study was to determine the performance characteristics of the Cozart Methadone Microplate ELISA assay for the detection of methadone and methadone metabolites in hair specimens. One hundred and ten hair specimens were collected from volunteers (n=46) with a history of drug use and from drug-related deaths (n=64). The hair samples (approximately 20 mg) were extracted by sonication in methanol followed by overnight extraction in methanol at 60 degrees C. The methanol extract was evaporated to dryness, reconstituted in ELISA negative calibrator, and then analyzed. For gas chromatography-mass spectrometry (GC-MS) analysis, deuterated internal standard mixture [methadone-d9 and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP)-d3] and 0.1M HCI were added to approximately 20 mg of specimen or spiked blank hair and sonicated for 1 h. The pH was adjusted to neutral, and methadone and its primary metabolite, EDDP, were analyzed by GC-MS following solid-phase extraction using Bond Elute Certify columns and pH 7.4 phosphate buffer (0.1M). Forty hair specimens were confirmed positive for methadone by GC-MS. Concentrations ranged from 0.10 to 8.3 ng/mg for methadone and 0.1 to 1.2 ng/mg for EDDP. The true positives, true negatives, false positives, and false negatives for different cutoffs with the ELISA were determined by comparison of the ELISA response (normalized to weight of hair extracted) to the GC-MS results with a cutoff of 0.1 ng/mg for both methadone and EDDP as the reference method. The optimum cutoff for the Cozart Methadone Microplate ELISA was determined to be between 200 and 300 pg methadone equivalents/mg hair using a 20 mg hair sample. The Cozart Methadone Microplate EIA for methadone and metabolites in hair using a cut-off of 200 pg/mg hair with a 20 mg hair sample had a sensitivity of 95 +/- 2% and a specificity of 100 +/- 3.5% (vs GC-MS) and an accuracy of 98.2 +/- 1.3%.

Comparison of GC-MS and EIA results for the analysis of methadone in oral fluid.

The purpose of these studies was to evaluate the performance characteristics of the Cozart Microplate Enzyme Immunoassay (EIA) for the determination of methadone in oral fluid from patients in a drug misuse treatment program. Oral fluid specimens were collected using the Cozart RapiScan Collection system from 198 donors who were receiving treatment for their addiction and were monitored for drug misuse. Oral fluid specimens were also collected from forty volunteer donors who were not drug users. The specimens were analyzed in the laboratory by EIA and then analysed for methadone and its main metabolite EDDP by gas chromatography-mass spectrometry (GC-MS). A total of 103 samples were confirmed positive for methadone. The Cozart Microplate EIA for d-Methadone in oral fluid using a cutoff of 30 ng/mL in diluted oral fluid had a sensitivity of 91.3% +/- 2.8% and a specificity of 100% +/- 1.0% vs. GC-MS.

Performance characteristics of the Cozart RapiScan Oral Fluid Drug Testing System for opiates in comparison to ELISA and GC/MS following controlled codeine administration.

Oral fluid is an interesting alternative matrix for drug testing in many environments, including law enforcement, workplace drug testing, and drug treatment facilities. Performance characteristics of the FDA-cleared, qualitative, Cozart RapiScan Opiate Oral Fluid Drug Testing System (Opiate Cozart RapiScan System or Opiate CRS) were compared to the semi-quantitative Cozart Microplate EIA Opiate Oral Fluid Kit (Opiate ELISA) and to gas chromatography/mass spectrometry (GC/MS). The following oral fluid opiate cutoffs were evaluated: the GC/MS limit of quantification (LOQ) of 2.5 mg/l; 15 microg/l currently used for oral fluid testing in the United Kingdom (UK); 30 microg/l (Opiate CRS cutoff); and 40 microg/l, the proposed Substance Abuse and Mental Health Services Administration (SAMHSA) cutoff. Subjects provided informed consent to participate in this IRB-approved research and resided on the closed research ward throughout the study. Three oral codeine doses of 60 mg/70 kg were administered over a 7-day period. After a 3-week break, subjects received three doses of 120 mg/70 kg within 7 days. Oral fluid specimens (N = 1273) were analyzed for codeine (COD), norcodeine (NCOD), morphine (MOR) and normorphine (NMOR) by GC/MS with an LOQ of 2.5 microg/l for all analytes. MOR and NMOR were not detected in any sample; 26.5% of the specimens were positive for COD and 13.7% for NCOD. Opiate CRS uses a preset, qualitative cutoff of 10 microg/l; this is equivalent to 30 microg/l in undiluted oral fluid as the oral fluid collection process involves a 1:3 dilution with buffer. Sensitivity, specificity, and efficiency of Opiate CRS compared to Opiate ELISA were 98.6, 98.1, and 98.2% at a 30 microg/l cutoff and 99.0, 96.2, and 96.6% at a 40 microg/l cutoff. Compared to the much lower GC/MS LOQ of 2.5 microg/l, sensitivity, specificity and efficiency were 66.8, 99.3 and 90.7%. Increasing the GC/MS cutoff to the current UK level yielded performance characteristics of 81.5% (sensitivity), 99.3% (specificity), and 95.4% (efficiency). Using a GC/MS cutoff identical to the preset Opiate CRS cutoff yielded sensitivity, specificity, and efficiency of 88.5, 99.2, and 97.5%, respectively. At the proposed SAMSHA confirmation cutoff of 40 microg/l, sensitivity increased with little change in specificity and efficiency (91.3% sensitivity, 98.9% specificity, and 97.5% efficiency). Oral fluid is a suitable matrix for detecting drugs of abuse. Opiate CRS, with a 30 microg/l cutoff, is sufficiently sensitive, specific and efficient for oral fluid opiate analysis, performing similarly to Opiate ELISA at the same cutoff, and having performance characteristics >91% when compared to GC/MS at the proposed SAMHSA cutoff.

Validation of the Cozart microplate ELISA for detection of opiates in hair.

The purpose of this study was to determine the performance characteristics of the Cozart Opiates Microplate ELISA assay for the detection of opiates in hair specimens. One hundred and six hair specimens were collected from volunteers and from drug-related deaths. The hair samples were extracted by sonication followed by overnight extraction in methanol at 60 degrees C. The methanol extract was dried, reconstituted in ELISA negative calibrator, and then analyzed. For gas chromatography-mass spectrometry (GC-MS) analysis, deuterated internal standard mixture and 0.1M HCl were added to 20 mg of specimen or spiked blank hair and sonicated for 1 h. The opiates were extracted by solid-phase and derivatized with BSTFA + 1% TMS for GC-MS analysis. Fifty-one hair specimens were confirmed positive by GC-MS. The true positives, true negatives, false positives, and false negatives for different cutoffs with the ELISA were determined by comparison of the ELISA response (normalized to weight of hair extracted) to the GC-MS results as the reference method. The optimum cutoff for the Cozart Opiate Microplate ELISA was determined to be between 200 and 300 pg morphine equivalents/mg hair using a 20-mg hair sample. The Cozart Opiates Microplate EIA for opiates in hair using a cutoff of 200 pg/mg hair with a 20-mg hair sample had a sensitivity of 98% +/- 2% and a specificity of 92.7% +/- 3.5% versus GC-MS.

Validation of the Cozart microplate EIA for cocaine and metabolites in oral fluid.

The purpose of these studies was to determine the performance characteristics of the Cozart microplate EIA for the detection of cocaine and cocaine metabolites in oral fluid. A total of 217 samples were collected using the Cozart RapiScan oral fluid Collection System from donors who were receiving treatment and being monitored for their addiction. The samples were screened in the laboratory using the Cozart microplate EIA for cocaine and metabolites and confirmed using gas chromatography-mass spectrometry (GC-MS). The samples were stored frozen until analysis by GC-MS. Of the 217 samples tested, 116 were positive for cocaine and/or cocaine metabolites. The Cozart microplate EIA for cocaine, using a cutoff of 30 ng/mL benzoylecgonine equivalents in neat oral fluid, had a sensitivity of 95.7% +/- 2.0% and specificity of 100% +/- 0.7%. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.

Cozart RapiScan Oral Fluid Drug Testing System: an evaluation of sensitivity, specificity, and efficiency for cocaine detection compared with ELISA and GC-MS following controlled cocaine administration.

Oral fluid has become a widely accepted alternative matrix for drugs of abuse detection. Immunoassays have been developed for on-site testing of cocaine and metabolites in oral fluid. The performance of the Cozart RapiScan Oral Fluid Drug Testing System (CRS) was evaluated in comparison with Cozart Microplate Enzyme Immunoassay Cocaine Oral Fluid Kit (COC ELISA) and gas chromatography-mass spectrometry (GC-MS) at several screening and confirmation cutoffs, including those proposed by SAMHSA and those currently in use in the U.K. Oral fluid samples (n = 1271) were collected prior to and following controlled clinical cocaine administration. CRS provides a qualitative screen at a preset cutoff of 30 microg/L. Sensitivity, specificity, and efficiency for CRS (30 microg/L) as compared with COC ELISA with a cutoff of 30 microg/L were 92.1%, 91.8%, and 92.0%. The comparison of CRS (30 microg/L) with the 8-mg/L proposed SAMHSA confirmation cutoffs for cocaine and/or benzoylecgonine exhibited a sensitivity of 82.7%, a specificity of 94.5%, and an efficiency of 87.6%. For this study, an alternative CRS cutoff of 20 microg/L was also evaluated. Performance characteristics of CRS (20 microg/L) at the proposed SAMHSA confirmation cutoffs were 89.9%, 89.7%, and 89.8%, respectively. At cutoffs in use in the U.K., 30- micro g/L CRS screen and 15- microg/L GC-MS cutoffs for cocaine, benzoylecgonine, and/or ecgonine methyl ester sensitivity, specificity, and efficiency were 89.4%, 92.2%, and 90.7%, respectively. Cozart RapiScan had performance similar to the COC ELISA assay for the detection of cocaine exposure and suitable sensitivity and specificity at the proposed SAMHSA cutoffs.

Effects of oral fluid contamination on two oral fluid testing systems.

Oral fluid is a popular matrix for drug testing; however, little has been published concerning the effect that food or beverages may exert on oral fluid screening tests. This study describes the effects of 19 different foods, beverages and vinegars on two test systems, the Concateno Certus and Orasure Intercept. Samples giving positive screening results were subjected to confirmatory analysis using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. Results showed that intermittent presumptive positive results for amphetamine, methadone, opiates and cocaine could be detected following the consumption of coffee, Coke, fruit juice, oranges, spicy food and toothpaste using the Orasure system if specimens were not collected in accordance with the manufacturer's recommended collection procedure. Following the consumption of vinegar, presumptive positives were observed using the Orasure system for up to 30 min post-exposure. No presumptive positives were observed using the Concateno system. It is a widely held view that foods and beverages disperse from the mouth within 10-15 min after their consumption, and hence are unlikely to affect oral fluid drug tests. This study shows that vinegar can affect immunoassay screening for an extended period following its consumption.