Mindy L. Rawlins

Performance Characteristics of Six Homocysteine Assays

Elevated concentrations of homocysteine (Hcy) are associated with a range of disorders. Linearity, imprecision, interference, method comparison, and accuracy were evaluated on the ADVIA Centaur (Siemens Healthcare Diagnostics, Deerfield, IL), ARCHITECT i2000SR (Abbott Diagnostics, Abbott Park, IL), AxSYM (Abbott Diagnostics), and IMMULITE 2000 (Siemens Healthcare Diagnostics) methods and analyzers and the Catch (Equal Diagnostics, Exton, PA) and Diazyme (Diazyme Laboratories, San Diego, CA) methods, both on the Modular P analyzer (Roche Diagnostics, Indianapolis, IN). All methods were linear with maximum deviations from target recoveries of less than 10%. Total coefficients of variation ranged from 1.7% to 9.4%. The effects of hemolysis, icterus, and lipemia were assessed. Method comparisons were performed using high-performance liquid chromatography as the comparison method. Correlation coefficients were 0.95 to 0.99. Bland-Altman plots demonstrated percentage bias of -29.3% (IMMULITE) to 7.2% (Centaur). Accuracy using the National Institute of Standards and Technology Standard Reference Material 1955 showed varying results with only 1 method within the certified range for all 3 levels. All methods demonstrated acceptable performance except the IMMULITE, which is less precise and accurate. Standardization of most methods seems acceptable, although continuing efforts are warranted.

Selected performance characteristics of the Roche Elecsys® testosterone II assay on the Modular analytics E 170 analyzer

BACKGROUND Serum testosterone measurements have utility in diagnosis of clinical conditions characterized by both increased and decreased testosterone concentrations. Studies have indicated that testosterone immunoassays may give inaccurate results for women and children. We evaluated the performance of a second generation testosterone immunoassay from Roche Diagnostics. METHODS Testo II performed on a Modular Analytics E 170 analyzer is an automated random access electrochemiluminometric assay. We evaluated limit of blank (LoB), imprecision, linearity, interference, and method comparison with liquid chromatography-tandem mass spectrometry assay (LC-MS/MS). Method comparison included the current generation Roche testosterone assay (Testo I) and the Access 2 testosterone chemiluminometric assay (Beckman Coulter). Results for men and women were analyzed for analytic concordance. The relative % differences of immunoassay compared to LC-MS/MS results were evaluated. RESULTS The LoB was 0.07nmol/l. Total imprecision was <6%. The assay was linear from 0.2 to 46.6nmol/l. Negative interference was observed for lipemia at concentrations >22.5g/l. Analytic concordance showed improved specificity for women. Comparison of results to LC-MS/MS indicated comparable performance with other immunoassays for men and improved performance for women, boys, and girls with mean differences of 0.5%, -0.7%, and 24.4%, respectively. CONCLUSIONS The Roche Testo II assay demonstrated excellent precision. Comparison to 2 other automated immunoassays showed comparable performance for men and improved performance for women and children. However, challenges still exist for quantifying testosterone concentrations <10.4nmol/l for men and <1.7nmol/l for women and children by immunoassay.

Evaluation of a Western Blot Method for the Detection of Yersinia Antibodies: Evidence of Serological Cross-Reactivity between Yersinia Outer Membrane Proteins and Borrelia burgdorferi

ABSTRACT Yersinia enterocolitica and Yersinia pseudotuberculosis have been identified as causative organisms of reactive arthritis in humans. We evaluated a Western blot assay which uses Yersinia outer membrane proteins as antigens for the detection of Yersinia antibodies as a replacement for the complement fixation (CF) assay. Clinical agreement, sensitivity, and specificity were determined by testing 19 positive and 21 negative serum samples by the CF assay, Western blot assay, and enzyme-linked immunosorbent assay (ELISA). The CF assay and ELISA were compared to the Western blot assay, which was the reference method used in this study. Sera with antibodies that could potentially cross-react with Yersinia were also tested by the Western blot assay. The agreement, sensitivity, and specificity of the CF method were 61%, 26%, and 95%, respectively; and those for the ELISA were 89%, 95%, and 82%, respectively. The prevalences of Yersinia antibodies in 50 healthy donors were 6% for immunoglobulin G (IgG), 2% for IgA, and 2% for IgM. Sera positive for Bartonella henselae, Brucella, Chlamydia pneumoniae, and Rickettsia rickettsii antibodies showed cross-reactivity by the Western blot assay. The highest cross-reactivity was observed with Borrelia burgdorferi; 5 of 11 (45%) specimens were cross-reactive by the IgM-specific assay. Overall, the Western blot assay performs acceptably and is more sensitive than the CF assay, warranting replacement of the CF assay in the laboratory. Due to the evidence of cross-reactivity, particularly with B. burgdorferi, which can cause an oligoarthritis similar to reactive arthritis, the diagnosis of reactive arthritis should be based on clinical findings and complete serologic analysis of the potential causative infectious pathogens.

Characterization of an Immunogenic Outer Membrane Autotransporter Protein, Arp, of Bartonella henselae

ABSTRACT Bartonella henselae is a recently recognized pathogenic bacterium associated with cat scratch disease, bacillary angiomatosis, and bacillary peliosis. This study describes the cloning, sequencing, and characterization of an antigenic autotransporter gene from B. henselae. A cloned 6.0-kb BclI-EcoRI DNA fragment expresses a 120-kDa B. henselae protein immunoreactive with 21.2% of sera from patients positive for B. henselae immunoglobulin G antibodies by indirect immunofluorescence, with 97.3% specificity and no cross-reactivity with antibodies against various other organisms. DNA sequencing of the clone revealed one open reading frame of 4,320 bp with a deduced amino acid sequence that shows homology to the family of autotransporters. The autotransporters are a group of proteins that mediate their own export through the outer membrane and consist of a passenger region, the α-domain, and an outer membrane transporter region, the β-domain. The passenger domain shows homology to a family of pertactin-like adhesion proteins and contains seven, nearly identical 48-amino-acid repeats not found in any other bacterial or Bartonella DNA sequences. The passenger α-domain has a calculated molecular mass of 117 kDa, and the transporter β-domain has a calculated molecular mass of 36 kDa. The clone expresses a 120-kDa protein and a protein that migrates at approximately 38 kDa exclusively in the outer membrane protein fraction, suggesting that the 120-kDa passenger protein remains associated with the outer membrane after cleavage from the 36-kDa transporter.

Evaluation of an Enzyme Immunoassay for Detection of Immunoglobulin M Antibodies to West Nile Virus and the Importance of Background Subtraction in Detecting Nonspecific Reactivity

ABSTRACT Since the introduction of West Nile virus (WNV) in the United States in 1999, several assays have become commercially available to detect antibodies against WNV. Capture enzyme-linked immunosorbent assays (ELISAs) for the detection of WNV-specific immunoglobulin M (IgM) have been approved by the Food and Drug Administration for clinical testing and are available from Focus Diagnostics and PanBio, Inc. The Focus Diagnostics IgM capture ELISA utilizes a background subtraction protocol in order to detect nonspecific reactivity due to rheumatoid factor, heterophile antibodies, or other interfering substances. A background subtraction procedure is not currently recommended for the PanBio IgM capture ELISA. In previous experiments, we determined the agreement, sensitivity, and specificity of the PanBio first-generation IgM capture ELISA compared to an immunofluorescence assay and the Centers for Disease Control and Preventions IgM capture ELISA. The PanBio assay has since been reformulated to improve the specificity of the assay. We evaluated the reformulated PanBio assay with and without an antigen subtraction procedure and compared the results to the Focus IgM capture ELISA. Agreement, sensitivity, and specificity of the PanBio assay were, respectively, 85%, 95%, and 76% without the subtraction protocol and 94%, 95%, and 93% with the subtraction protocol. In general, when the subtraction protocol was applied to the PanBio IgM capture ELISA, there was a reduction in some, but not all, false-positive results. We suggest that all WNV IgM assays be standardized with a procedure such as background subtraction to eliminate nonspecific reactivity that may cause false-positive results.

Performance characteristics of the Access® Inhibin A assay

BACKGROUND Second trimester maternal screening using AFP, uE3, hCG, and inhibin A has shown a detection rate for Downs syndrome of 81% with a 5% false positive rate. Inhibin A may also have utility as a serum tumor marker in postmenopausal women with ovarian cancer and men with testicular stromal tumors. METHODS The Beckman Coulter Access Inhibin A assay was evaluated for limit of blank, dilution linearity, imprecision, interferences, reference intervals, and comparison to an inhibin A ELISA. RESULTS The limit of blank was 0.1 ng/l. The assay was linear from 0.2 to 1347 ng/l. Total inter-assay CVs were <5% for control levels ranging from 24.6 ng/l to 811 ng/l. Interference studies showed recoveries of inhibin A within 10% of expected values at interferent concentrations of 10 g/l hemoglobin and 22 g/l triglycerides. No significant interference was observed at a bilirubin concentration of 400 mg/l. The 97.5th percentile upper reference limits were 6.8 ng/l for postmenopausal women and 3.0 ng/l for men. The Access assay compared to an ACTIVE ELISA showed a slope of 0.88, an intercept of -3.7 ng/l, S(y/x)=40 ng/l, and r=0.98. CONCLUSIONS The analytical performance of the Access inhibin A assay is acceptable for routine laboratory testing.

Performance characteristics of an immunoturbidimetric assay for lipoprotein-associated phospholipase A2.

BACKGROUND Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is an inflammatory biomarker for coronary heart disease and ischemic stroke risk assessment. METHODS The Plac turbidimetric immunoassay (TIA) for measurement of Lp-PLA(2) was evaluated for limit of blank, functional sensitivity, dilution linearity, imprecision, interferences, and comparison to an ELISA assay for Lp-PLA(2). RESULTS The assay was linear from 100 to 500 microg/l. Total inter-assay CVs were <3% for both control levels. Interference studies showed recoveries of 90-110% of expected values at interferent concentrations up to 15 g/l hemoglobin, 450 mg/l bilirubin, and 41 g/l triglycerides. Comparison of 3 sample sets collected using different protocols gave the following regression statistics for the Plac TIA compared to the Plac ELISA: sample set 1 (n=99) slope=1.5, intercept=-122.4, S(y/x)=71.3 microg/l, and r=0.61; sample set 2 (n=100) slope=2.1, intercept=-173 microg/l, S(y/x)=55 microg/l, and r=0.82; sample set 3 after 1 freeze-thaw cycle (n=30) slope=1.2, intercept=-68.6 microg/l, S(y/x)=28.6 microg/l, and r=0.82; sample set 3 after a second freeze-thaw cycle slope=1.4, intercept=-68.2 microg/l, S(y/x)=33.1 microg/l, and r=0.83. CONCLUSIONS The Plac TIA reagents perform acceptably on the Roche Modular P analyzer. However, only a fair correlation was observed between the Plac ELISA and Plac TIA assays.