Inter-assay Precision Studies Research Articles

Analytical Validation of NavDx, a cfDNA-Based Fragmentomic Profiling Assay for HPV-Driven Cancers

The NavDx® blood test analyzes tumor tissue modified viral (TTMV)-HPV DNA to provide a reliable means of detecting and monitoring HPV-driven cancers. The test has been clinically validated in a large number of independent studies and has been integrated into clinical practice by over 1000 healthcare providers at over 400 medical sites in the US. This Clinical Laboratory Improvement Amendments (CLIA), high complexity laboratory developed test, has also been accredited by the College of American Pathologists (CAP) and the New York State Department of Health. Here, we report a detailed analytical validation of the NavDx assay, including sample stability, specificity as measured by limits of blank (LOBs), and sensitivity illustrated via limits of detection and quantitation (LODs and LOQs). LOBs were 0-0.32 copies/μL, LODs were 0-1.10 copies/μL, and LOQs were <1.20-4.11 copies/μL, demonstrating the high sensitivity and specificity of data provided by NavDx. In-depth evaluations including accuracy and intra- and inter-assay precision studies were shown to be well within acceptable ranges. Regression analysis revealed a high degree of correlation between expected and effective concentrations, demonstrating excellent linearity (R2 = 1) across a broad range of analyte concentrations. These results demonstrate that NavDx accurately and reproducibly detects circulating TTMV-HPV DNA, which has been shown to aid in the diagnosis and surveillance of HPV-driven cancers.

Analytical and clinical validation of an RNA sequencing-based assay for quantitative, accurate evaluation of a molecular signature response classifier in rheumatoid arthritis

ABSTRACT Objectives This study reports the analytical and clinical validation of a molecular signature response classifier (MSRC) that identifies rheumatoid arthritis (RA) patients who are non-responders to tumor necrosis factor-ɑ inhibitors (TNFi). Methods The MSRC integrates patient-specific data from 19 gene expression features, anti-cyclic citrullinated protein serostatus, sex, body mass index and patient global assessment into a single score. Results MSRC results stratified samples (N=174) according to signature of non-response detection with a positive predictive value of 87.7% (95% CI: 78-94%), sensitivity of 60.2% (95% CI: 50-69%) and specificity of 77.3% (95% CI: 65-87%). The continuous 25-point scale was sub-divided into three thresholds: signal not detected (<10.6), high (≥10.6) and very high (≥18.5). The MSRC relies on sequencing of RNA extracted from blood; this assay displayed high gene expression concordance among inter- and intra-assay sample pairs (R2 > 0.977) and minimal variation in cumulative gene assignment diversity, read mapping location or gene-body coverage. The MSRC had an accuracy of 95.8% (46/48) for threshold concordance (no signal, high, very high). Intra- and inter-assay precision studies demonstrated the MSRC had high repeatability (92.6%, 25/27) and reproducibility (100%, 35/35). Conclusion The MSRC is a robust assay that accurately and reproducibly detects an RA patient’s molecular signature of non-response to TNFi therapies.

Implementation of a Fully Automated Latex Immunoturbidimetric Assay for the Diagnosis of Heparin-Induced Thrombocytopenia in a High-Volume University Hospital Laboratory

Abstract Background Heparin-induced thrombocytopenia (HIT) is an immune-mediated, adverse reaction to heparin in which heparin binds platelet factor 4 (PF4), triggering the development of heparin-PF4 antibodies (HITAb). HITAbs bind and activate platelets, causing thrombosis, platelet consumption, and thrombocytopenia. Heparin is replaced with relatively costly nonheparin anticoagulants until HIT can be ruled out. HIT diagnosis consists of a HITAb immunoassay with reflex to a serotonin release assay (SRA) for confirmation of positive results. Recently, a fully automated latex immunoturbidimetric assay (LIA) for detection of HITAbs received FDA clearance. We sought to verify the performance characteristics of the LIA with the aim of implementing the test in a high-volume university hospital laboratory. Methods The in-house HITAb LIA was performed on the Instrumentation Laboratory TOP700 analyzer using HemosIL HIT-Ab(PF4-H) reagent. The comparator method, a HITAb ELISA, was performed at a reference laboratory with positive results reflexed to SRA. All samples (36 total) sent to the reference laboratory for HIT testing from December 2017 to November 2018 were aliquoted and run in parallel by LIA. Intra-assay precision was assessed by running manufacturer-provided low and high control samples 10 times in succession, while interassay precision was assessed by running low and high samples every day for 10 days. Turnaround time to HITAb result was retrieved from the electronic medical records for HIT testing performed 60 days before and after in-house test implementation. Results The agreement between the LIA and ELISA was 92% (33/36). One discordant sample tested negative by ELISA and was not assessed by SRA. Another tested positive by ELISA and negative by LIA and was confirmed negative by SRA. The final discordant sample tested negative by LIA but positive by ELISA and was confirmed positive by SRA. Thirty-three percent (12/36) of samples tested positive for HITAb by ELISA and were reflexed to SRA. Both the ELISA and LIA showed 83% agreement (10/12) with the SRA. The coefficient of variance (CV) for the intra-assay precision studies was 18% and 5% for the low and high controls, respectively. The CV for the interassay precision studies was 28% and 5% for the low and high controls, respectively. Postimplementation quality control data revealed 61% and 20% imprecision on the low and high level controls, respectively, which declined significantly when reagents were removed from the instrument and refrigerated within 2 hours. The turnaround time for HITAb results was reduced by 74% (10.5 vs 41 hours) after in-house test implementation, significantly reducing the need for administration of nonheparin anticoagulants. Conclusion The LIA and ELISA methods compared favorably, allowing for clinical implementation of the LIA. The shortened turnaround time of the LIA significantly reduced the time to rule out HIT, enhancing patient care and reducing drug costs. The assay imprecision warrants further investigation regarding reagent stability.

Analytical validation of a 47-gene NGS panel for molecular profiling of myeloid neoplasms.

e18526 Background: Molecular profiling can help diagnose, classify, and guide treatment of myeloid neoplasms. Next generation sequencing (NGS) is a powerful platform that can facilitate identification of actionable genetic alterations in multiple genes simultaneously. To serve clinical management needs, we developed and validated a NGS panel for 47 genes associated with diagnostic, prognostic, and/or therapeutic utilities for each of the subclasses of myeloid neoplasms: acute myeloid leukemia (AML, 42 genes), myelodysplastic syndrome (MDS, 36 genes), and myeloproliferative neoplasms (MPN, 26 genes). Methods: A total of 154 unique de-identified specimens were analyzed. Intra- and inter-assay precision studies were performed by standard laboratory practice. Single nucleotide variations (SNVs), insertions/deletions (indels), and FLT3 partial tandem duplications (PTD) were assessed for the entire coding regions of 23 genes and targeted exons of 24 genes. Target regions were captured by hybridization with complementary biotinylated DNA baits, and NGS was performed on an Illumina NextSeq500 instrument. Sequence reads obtained were analyzed using a bioinformatics pipeline that was developed in-house. At least 500 unique reads with base quality (Q) ≥20 (99% confidence) were required for coverage QC, except targets on X chromosome for male specimens, which required 250 unique reads. Results: On average, each sample generated 13.2 million reads, 100% (SD = 0.2%) mapped reads to the reference sequence, 61.7% (SD = 8.8%) on-target reads, and 1,952 (SD = 873) mean coverage. Data using ≥Q20 unique coverage of all target regions across all validation samples demonstrated that all target regions, even in the extreme end of GC spectrum (range: 12%-87%), met coverage requirements. Intra- and inter-assay precision studies, using 7 and 21 specimens, respectively, resulted in 100% concordance among replicates. An accuracy study of 131 unique specimens (a subset of the 154 specimens) yielded in 99.6% (755/758, 95% CI: 99.4%-100%) concordance for variants with allele frequencies ≥5% in comparison to other molecular methods including Sanger sequencing. No reportable variants were detected from any normal specimens (n = 17). Conclusions: We developed and validated a NGS panel for 47 genes with diagnostic, prognostic, and/or therapeutic utilities for each of the subclasses of myeloid neoplasms: AML (42 genes), MDS (36 genes), and MPN (26 genes). This panel will allow simultaneous analysis of the 47 genes with high sensitivity and specificity and may help manage clinical needs.

Clinical validation of long-range PCR assay for MLL partial tandem duplications for acute myeloid leukemia.

7033 Background: Partial tandem duplication of the mixed lineage leukemia gene ( MLL-PTD) occurs in about 3% to 5% of adult acute myeloid leukemia (AML), mostly in patients with normal karyotypes, and is associated with poor prognosis. Real-time PCR on cDNA or RT-PCR followed by gel electrophoresis are the most commonly used methods for the detection of the mutation. However, those methods require RNA extraction, which is not often performed for molecular cytogenetic testing or next generation sequencing (NGS) for AML. In this study, we developed an MLL-PTD detection assay that uses long-range PCR on extracted DNA, a commonly processed sample type for AML molecular testing, to reduce sample processing requirements. Methods: DNA was extracted from de-identified 72 whole blood and 30 bone marrow aspirate specimens. Two PCR reactions were performed for each DNA sample: one to detect MLL-PTD and another to detect CHEK2 to monitor DNA integrity. The 3 most frequent forms of MLL-PTD (exons 2~8, 2~9, and 2~10) were detected using a forward DNA primer on exon 8 and a reverse primer on exon 2. MLL-PTD positivity was determined by the intensity of amplified DNA fragment at or above the lower detection limit control of this assay. Intra-assay precision, inter-assay precision, and limit of detection (LOD) were performed according to standard laboratory protocols. Results of this method were compared to those of NGS. Results: Intra-assay precision studies on 5 specimens yielded 100% genotype concordance. Inter-assay precision studies on 20 specimens yielded 100% concordance between replicates. LOD studies using a human eosinophilic leukemia cell line (EoL-1) with 12.5-kb PTD in size showed assay sensitivity of 5%; for smaller MLL-PTD positives (PTD ~3 kb and ~5 kb), LOD was 1%. For method comparison studies, 71 specimens (52 whole blood and 19 bone marrow) were also analyzed by NGS; concordance between the methods was 97.2% (69/71). Conclusions: We developed and validated long-range MLL-PTD detection assay for AML. The developed method uses DNA, which is commonly used for molecular testing for AML, and thus may reduce sample processing time by consolidating sample type across tests.

1996. Addressing Pulmonary Nocardiosis Risk in Immunocompromised Patients: Development and Validation of a Commercially Available PCR

BackgroundPulmonary nocardiosis is an infection targeting immunocompromised patients characterized by high mortality and requires nonfrontline antibiotics for treatment. Nocardiosis is currently confirmed or excluded by BAL fluid culture followed by further phenotypic identification steps. A culture-independent method with more timely results would accelerate the administration of appropriate treatment. A rapid Nocardia (NOC) PCR assay for BAL has neither been previously validated nor offered for clinical testing to our knowledge.MethodsOligonucleotides for a rapid NOC PCR comprehensive of the causative agents of nocardiosis were aligned to the 16S regions of common NOC species and over 30 others. Specificity was verified against publically available bacterial 16S sequences. Rapid automated nucleic acid extraction (<1 hour for 24 samples) followed by fast PCR (<1 hour) was validated according to relevant compliance standards. Spiked/unspiked human BAL samples were used to assess analytical specificity, limit of detection (LOD), precision and accuracy using NOC and non-NOC strains.ResultsThe NOC PCR detected, among others, the most common NOC species (N. cyriacigeorgica, N. nova, N. farcinica and N. brasiliensis). We estimate more than 95% of causative agents of nocardiosis are detectable by the assay. No cross reactivity was detected from 30 non-NOC bacterial pathogens except for Rhodococcus and Crossiella spp. LOD in BAL fluid was determined to be 206, 41, and 26 copies/mL for N. cyriacigeorgica, N. nova, and N. transvalensis, respectively. Intra- and inter-assay precision studies revealed copies/mL %CV’s of <10% and <8% at a high concentration and <21% and <26% at a low concentration, respectively. Accuracy studies yielded 100% concordance with 33 BAL positives and 20 BAL negatives.ConclusionThe specificity, inclusivity, sensitivity, precision and accuracy of a qualitative PCR have been deployed as an aid in the diagnosis of pulmonary nocardiosis. NOC PCR allows for a culture-independent method that can rapidly detect clinically relevant NOC species with an improved turnaround time, leading to prompt diagnosis and administration of appropriate treatment.Disclosures E. Smith, Viracor Eurofins Clinical Diagnostics: Employee, Salary. K. Bartlett, Viracor Eurofins Clinical Diagnostics: Employee, Salary. J. Grantham, Viracor Eurofins Clinical Diagnostics: Employee, Salary. N. Powell, 1987: Employee, Salary. M. Altrich, Viracor Eurofins Clinical Diagnostics: Employee, Salary. S. Kleiboeker, Viracor Eurofins Clinical Diagnostics: Employee, Salary. M. Wissel, Viracor Eurofins Clinical Diagnostics: Employee, Salary.

Dispersive liquid-liquid microextraction of quinolones in porcine blood: Validation of a CE method using univariate calibration or multivariate curve resolution-alternating least squares for overlapped peaks.

In the previously published part of this study, we detailed a novel strategy based on dispersive liquid-liquid microextraction to extract and preconcentrate nine fluoroquinolones in porcine blood. Moreover, we presented the optimized experimental conditions to obtain complete CE separation between target analytes. Consequently, this second part reports the validation of the developed method to determine flumenique, difloxacin, enrofloxacin, marbofloxacin, ofloxacin, ciprofloxacin, through univariate calibration, and enoxacin, danofloxacin, and gatifloxacin through multivariate curve resolution analysis. The validation was performed according to FDA guidelines for bioanalytical assay procedures and the European Directive 2002/657 to demonstrate that the results are reliable. The method was applied for the determination of fluoroquinolones in real samples. Results indicated a high selectivity and excellent precision characteristics, with RSD less than 11.9% in the concentrations, in intra- and interassay precision studies. Linearity was proved for a range from 4.00 to 30.00 mg/L and the recovery has been investigated at four different fortification levels, from 89 to 113%. Several approaches found in the literature were used to determinate the LODs and LOQs. Though all strategies used were appropriate, we obtained different values when using different methods. Estimating the S/N ratio with the mean noise level in themigration time of each fluoroquinolones turned out as the best studied method for evaluating the LODs and LOQs, and the values were in a range of 1.55 to 4.55 mg/L and 5.17 to 9.62 mg/L, respectively.

Development of a high-throughput β-Gal-based neutralization assay for quantitation of herpes simplex virus-neutralizing antibodies in human samples

Measurement of neutralizing antibodies against herpes simplex virus (HSV) is important for evaluation of candidate vaccines. The established plaque-reduction neutralization assay is time consuming, labor intensive, and difficult to validate and transfer. Here, we describe the characterization of a HSV-neutralization assay based on the expression of a reporter gene, β-galactosidase (β-Gal). Using previously constructed HSV-β-Gal recombinant viruses, HSV-2/Gal and HSV-1/tk12, we developed a colorimetric β-Gal-based neutralization assay that is sensitive and highly reproducible, and performed in less than 48h. HSV-1 and HSV-2 neutralizing titers measured by the β-Gal-based neutralization assay were equivalent to those obtained by a plaque reduction neutralization assay. Intra- and inter-assay precision studies demonstrated that the β-Gal-based assay was repeatable and yielded low and acceptable variation. In addition, comparison of HSV-2 neutralizing antibody (NAb) titers measured in two independent laboratories by two unique β-Gal-based assays showed a highly significant correlation (r=0.9499, p<0.0001) between the two assays. The new assay will serve as an important tool both for preclinical and clinical trials of new HSV vaccines.

Enhanced high-performance liquid chromatography method for the determination of retinoic acid in plasma. Development, optimization and validation

When determining endogenous compounds in biological samples, the lack of blank or analyte-free matrix samples involves the use of alternative strategies for calibration and quantitation. This article deals with the development, optimization and validation of a high performance liquid chromatography method for the determination of retinoic acid in plasma, obtaining at the same time information about its isomers, taking into account the basal concentration of these endobiotica. An experimental design was used for the optimization of three variables: mobile phase composition, flow rate and column temperature through a central composite design. Four responses were selected for optimization purposes (area under the peaks, quantity of peaks, analysis time and resolution between the first principal peak and the following one). The optimum conditions resulted in a mobile phase consisting of methanol 83.4% (v/v), acetonitrile 0.6% (v/v) and acid aqueous solution 16.0% (v/v); flow rate of 0.68mLmin−1 and an column temperature of 37.10°C. Detection was performed at 350nm by a diode array detector. The method was validated following a holistic approach that included not only the classical parameters related to method performance but also the robustness and the expected proportion of acceptable results lying inside predefined acceptability intervals, i.e., the uncertainty of measurements. The method validation results indicated a high selectivity and good precision characteristics that were studied at four concentration levels, with RSD less than 5.0% for retinoic acid (less than 7.5% for the LOQ concentration level), in intra and inter-assay precision studies. Linearity was proved for a range from 0.00489 to 15.109ngmL−1 of retinoic acid and the recovery, which was studied at four different fortification levels in phuman plasma samples, varied from 99.5% to 106.5% for retinoic acid. The applicability of the method was demonstrated by determining retinoic acid and obtaining information about its isomers in human and frog plasma samples from different origins.

A novel ion-pairing chromatographic method for the simultaneous determination of both nicarbazin components in feed additives: Chemometric tools for improving the optimization and validation

The development, optimization and validation of an ion-pairing high performance liquid chromatography method for the simultaneous determination of both nicarbazin (NIC) components: 4,4′-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine (HDP) in bulk materials and feed additives are described. An experimental design was used for the optimization of the chromatographic system. Four variables, including mobile phase composition and oven temperature, were analyzed through a central composite design exploring their contribution to analyte separation. Five responses: peak resolutions, HDP capacity factor, HDP tailing and analysis time, were modelled by using the response surface methodology and were optimized simultaneously by implementing the desirability function. The optimum conditions resulted in a mobile phase consisting of 10.0mmolL−1 of 1-heptanesulfonate, 20.0mmolL−1 of sodium acetate, pH=3.30 buffer and acetonitrile in a gradient system at a flow rate of 1.00mLmin−1. Column was an INERSTIL ODS-3 (4.6mm×150mm, 5μm particle size) at 40.0°C. Detection was performed at 300nm by a diode array detector. The validation results of the method indicated a high selectivity and good precision characteristics, with RSD less than 1.0% for both components, both in intra and inter-assay precision studies. Linearity was proved for a range of 32.0–50.0μgmL−1 of NIC in sample solution. The recovery, studied at three different fortification levels, varied from 98.0 to 101.4 for HDP and from 99.1 to 100.2 for DNC. The applicability of the method was demonstrated by determining DNC and HDP content in raw materials and commercial formulations used for coccidiosis prevention. Assays results on real samples showed that considerable differences in molecular ratio DNC:HDP exist among them.

Evaluation of a New Rapid and Fully Automated Electrochemiluminescence Immunoassay for Thyrotropin Receptor Autoantibodies

Hyperthyroidism in Graves' disease is caused by autoantibodies to the TSH receptor (TSHR), and measurement of the TSHR autoantibody (TRAb) yields important information to diagnose and decide on the course of treatment of Graves' disease. We evaluated basic and clinical performance of a new, rapid, and fully automated electrochemiluminescence immunoassay Elecsys Anti-TSHR (Elecsys TRAb) for measuring serum TRAb. For evaluation of basic performance of the assay, we carried out intra- and interassay precision studies using five serum pools and three serum pools, respectively, and the assay was compared with four commercial TRAb assays. Clinical performance of the assay was evaluated with sera from 298 patients with untreated Graves' disease, 220 patients with destructive (painless and subacute) thyroiditis, and 332 healthy volunteers. The optimal cutoff point, which was calculated by receiver operating characteristic (ROC) analysis with the above subjects, was then used to classify an independent sample set of 80 patients with untreated Graves' disease, and 152 patients with destructive thyroiditis. Intraassay coefficient of variation (CV) was 4.24% at 1.85 IU/L and interassay CV was 10.1% at 1.46 IU/L. All the correlation coefficient values calculated against four commercial assays were larger than 0.85. ROC analysis resulted in a specificity of 99.1% with a sensitivity of 97.0% at a decision limit of 1.86 IU/L from comparison with untreated Graves' disease and destructive thyroiditis. The cutoff point yielded a sensitivity of 87.5% and specificity of 96.7% with the independent sample set. In spite of the short measuring time of only 27 minutes, the assay showed the same or better results with the existing commercial products. The short measuring time would contribute to speedy, preconsultation diagnosis of thyroid disease, especially of Graves' disease.

Latex Immunoturbidimetric Assay for Soluble Fibrin Complex

Soluble fibrin complex (SFC), composed of fibrin monomer and fibrinogen derivatives, is known to exist in the circulating blood in patients with thrombosis. Its detection and quantification are useful for obtaining information about the condition and degree of intravascular coagulation in early-stage thrombosis, but there is no rapid method to measure SFC in plasma for clinical use. We obtained a monoclonal antibody that specifically reacts with SFC, with desAA-fibrin as the immunogen, and developed a rapid and sensitive latex immunoturbidimetric assay (LIA) using latex-immobilized anti-SFC monoclonal antibody. The assay system was based on the increase in turbidity induced by the reaction of the latex-immobilized anti-SFC monoclonal antibody with SFC in plasma, and the assay procedure was fully automated on a Hitachi 911 analyzer. The method had an analytical range of 3-300 mg/L. Intra- and interassay precision studies indicated that this system provided reproducible data (CVs <3.0% and <2.0%, respectively). The assay detection limit was <0.5 mg/L. There was no interference from bilirubin (up to 440 mg/L), hemoglobin (up to 9.6 g/L), Intralipid (up to 10%), D-dimer (up to 200 mg/L), and rheumatoid factor (up to 470 000 IU/L). SFC concentrations in plasma from patients with thrombotic diseases [mean (SD), 48.9 (57.6) mg/L; n = 160) were significantly higher than those in plasma from healthy individuals [1.8 (2.1) mg/L; P <0.001; n = 304]. In terms of linearity, precision, and sensitivity, the LIA, performed on a Hitachi 911 automated analyzer, may be useful for measurement of SFC in plasma.

A novel monoclonal antibody to fibrin monomer and soluble fibrin for the detection of soluble fibrin in plasma

Background: Soluble fibrin (SF), composed of fibrin monomer (FM) and fibrinogen, is well known to exist in the circulating blood derived from patients with thrombotic diseases, and its quantification is useful to get some information on the state and degree of intravascular coagulation. However, there was no convenient method for the determination of SF. Methods: We prepared a novel monoclonal antibody (MoAb) (F405) to FM and SF using desAA-fibrin as the immunogen in the presence of anti-polymerant peptide (Gly-Pro-Arg-Pro, GPRP), and the characterization of the F405 was performed by Western blotting analysis and an enzyme-linked immunosorbent assay (ELISA). We also tried to detect SF in human plasma using an ELISA involving the immobilized F405 and horseradish peroxidase (POD)-labeled anti-fibrinogen polyclonal antibody. Results: The antibody reacted with the fibrin degradation products fragments X, Y and E, but not with fibrinogen or its fragments X, Y, D and E, or the fibrin D-dimer. The epitope recognized by F405 appeared to be the α-chain N-terminal region exposed upon removal of the A peptide from the Aα-chain because F405 was found to bind to the α-chain N-terminal oligo-peptide of fibrin (GPRVVERHQ). Since F405 reacted not only with FM in the presence of GPRP peptide, but also with the SF complex prepared by the addition of thrombin-treated FM to human fibrinogen, we attempted to detect SF in human plasma using ELISA. The analytical range of this method was 1–300 μg/ml. The assay detection limit was <0.5 μg/ml, and the results of intra- and inter-assay precision studies indicated that this method is accurate and yields reproducible results (<9.4% and <10%, respectively). When 56 samples of plasma from patients with disseminated intravascular coagulation (DIC) and 117 control samples from healthy individuals were tested, elevated levels of SF complex were detected in the DIC samples: the mean±S.D. of the SF concentration in the DIC and control samples were 63.4±65.3 μg/ml and 1.9±1.0 μg/ml, respectively. Conclusions: The ELISA using F405 is useful for the diagnosis of DIC.

High performance liquid chromatographic assay for the quantitation of total glutathione in plasma

A simple and widely used homocysteine HPLC procedure was applied for the HPLC identification and quantitation of glutathione in plasma. The method, which utilizes SBDF as a derivatizing agent utilizes only 50 μl of sample volume. Linear quantitative response curve was generated for glutathione over a concentration range of 0.3125–62.50 μmol/l. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.999. Limit of detection (LOD) and limit of quantitation (LOQ) values were 5.0 and 15 pmol, respectively. Glutathione recovery using this method was nearly complete (above 96%). Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of glutathione was demonstrated successfully using human and rat plasma samples.

VALIDATION OF A SOLILD PHASE EXTRACTION PROCEDURE FOR THE GC-MS IDENTIFICATION AND QUANTITATION OF COCAINE AND THREE METABOLITES IN BLOOD, URINE, AND MILK

A simple and widely used solid-phase extraction procedure (United Chemical Technologies Method Handbook) was applied for the GC-MS identification and quantitation of cocaine (COC), benzoylecgonine (BE), cocaethylene (COCE), and m-hydroxy- benzoylecgonine (HBE) in blood, urine, and milk. The method which utilizes BSTFA as a derivatizing agent yielded abundant diagnostic ions with high m/z values. Linear quantitative response curves were generated for the analytes of interest over a concentration range of 5–1000 ng/mL. Linear regression analyses of the standard curve in the three specimen types exhibited correlation coefficients ranging from 0.997 to 1.000. The LOD values for COC, COCE, and derivatives of BE, and HBE in the three specimen types ranged from 2.5 to 5.0 ng/mL. The LOQ values, however, ranged from 5.0 to 10.0 ng/mL. Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the detection and quantitation of COC, BE, COCE, and HBE was demonstrated successfully in human blood and urine samples, as well as blood samples obtained from cocaine-treated (subcutaneously) rats.

Evaluation of an automated, homogeneous enzyme immunoassay for serum thyroxine measurement in dog and cat serum.

A homogenous enzyme immunoassay (EIA) for measurement of serum thyroxine (T4) concentration was evaluated for use with canine and feline serum. The EIA method was linear from 0 to 150 nmol T4/L for human serum, 0 to 94 nmol T4/L for feline serum and 10 to 60 nmol T4/L for canine serum. Intra- and interassay precision studies yielded coefficients of variation </= 8% using single point measurements. Method comparison studies gave close agreement between radioimmunoassay (RIA) and EIA results. Correlation coefficients (r values) were 0.88 and 0.97 for canine and feline samples respectively, after application of species-specific factors to correct the calibrator values assigned for human serum samples. The EIA showed no interference from hemolysis at hemoglobin concentrations </= 20 g/L or from moderate lipemia. Highly lipemic specimens could be tested after centrifugation to clarify the sample. The EIA showed less interference from autoantibodies to T4 than the RIA method. The EIA method allows automation of T4 testing in a veterinary hospital or laboratory, and can be integrated with the routine clinical chemistry panel.

Quantitative determination of Orlistat (tetrahydrolipostatin, Ro 18-0647) in human plasma by high-performance liquid chromatography coupled with ion spray tandem mass spectrometry.

A rapid, sensitive and specific analytical method was developed and validated to quantify tetrahydrolipostatin (Orlistat, Ro 18-0647) in human plasma in order to provide pharmacokinetic data from clinical trials. This method employs a preliminary plasma protein precipitation step followed by a simple, one-step liquid-liquid extraction procedure to isolate Ro 18-0647 and its pentadeuterated internal standard, Ro 18-0647-d5, from the biological matrix. Reconstituted extracts were analyzed by liquid chromatography/ion spray tandem mass spectrometry (LC/MS/MS) in the selected reaction monitoring (SRM) mode. Chromatography was carried out using a 2 mm i.d. x 50 mm Deltabond Phenyl column. The eluent was acetonitrile-2 mM ammonium acetate (90:10). The retention time of the analyte was 1.2 min and chromatographic run times were less than 1.5 min. No interferences from anticoagulants, collection devices or endogenous constituents of the plasma were observed. The assay has a lower limit of quantitation (LLQ) of 0.20 ng ml-1 in plasma and a lower limit of detection (LLD) of 0.10 ng ml-1 plasma, based on 1 ml aliquots. The capability to detect 0.025 ng ml-1 in plasma has also been demonstrated. The calibration graphs were linear from 0.20 to 10 ng ml-1. The assay was initially validated with a linear range of 0.20-1.0 ng ml-1. This range was later extended and validated to an upper level of quantitation of 10 ng ml-1. Intra- and inter-assay precision studies showed a mean variability of less than 10%. The recovery, inter-assay precision and accuracy of the method were within acceptable bioanalytical standards. The assay has been shown to reliably provide automated, unattended sample analysis for approximately 150 samples per day. In an additional series of tests, Ro 18-0467 was shown to be stable under conditions that might be encountered during the analysis of samples from clinical trials. This LC/MS/MS assay procedure for Ro 18-0647 in human plasma has proven to be robust, sensitive, specific, accurate and reproducible. This method has been used to analyze over 5000 study samples.

Latex agglutination test for detecting antibodies to Treponema pallidum

We have developed an automated latex agglutination test for Treponema pallidum (TPLA) for measurement of the concentration of treponemal antibodies in syphilitic sera. The assay needs neither a complicated pretreatment of the sera nor special techniques. Intra- and interassay precision studies showed high reproducibility. Potentially interfering substances, such as bilirubin, hemoglobin, triglycerides, and rheumatoid factor, did not affect the results. The results obtained with the TPLA test showed a strong correlation with those from conventional methods. This test for antibodies to T. pallidum may be useful for the mass screening of syphilitic infections.

Comparison of the Vitek Immunodiagnostic Assay System with three immunoassay systems for detection of cytomegalovirus-specific immunoglobulin G

The Vitek Immunodiagnostic Assay System (VIDAS; bioMerieux Vitek Inc., Hazelwood, Mo.) was evaluated for its ability to detect anticytomegalovirus immunoglobulin G (IgG) and was compared with the following assay systems: Abbott IMx Cytomegalovirus IgG antibody assay (Abbott Laboratories, Abbott Park, Ill.), Whittaker Cytomegelisa II IgG assay (Whittaker Bioproducts, Walkersville, Md.), and Whittaker FIAX Cytomegalovirus IgG assay (Whittaker Bioproducts). Samples were considered positive if at least two of three (IMx, Cytomegelisa II, and FIAX) comparative assays gave positive results; a sample was considered negative if at least two of the three assays were negative. Of the 199 clinical serum samples tested, 194 gave concordant results among the three comparative assays (145 were positive and 49 were negative). The VIDAS results with two samples were persistently equivocal and were therefore excluded from the comparison study. The overall agreement between VIDAS and the established standard was 98.5%. The VIDAS assay yielded one false-positive and two false-negative results. The intra- and interassay precision studies indicate that the VIDAS assay is suitable for clinical use. The VIDAS system is rapid, self-contained, and fully automated and would be useful for screening of cytomegalovirus immune status.