Biomedical subjects

Elizabeth Valentine-Thon

Publications and source records attributed to Elizabeth Valentine-Thon.

6 recordsLinked to original sources

PubMed2006

A novel lymphocyte transformation test (LTT-MELISA) for Lyme borreliosis.

Diagnosis of active Lyme borreliosis (LB) remains a challenge in clinically ambiguous, serologically indeterminant, and polymerase chain reaction-negative patients. Lymphocyte transformation tests (LTTs) have been applied to detect specific cellular immune reactivity, but their clinical application has been severely hampered by the poorly defined Borrelia antigens and nonstandardized LTT formats used. In this study, we describe the development and clinical relevance of a novel LTT using a validated format (MELISA) together with well-defined recombinant Borrelia-specific antigens. From an initial screening of 244 patients with suspected Borrelia infection or disease, 4 informative recombinant antigens were selected: OspC (Borrelia afzelii), p41-1 (Borrelia garinii), p41-2 (B. afzelii), and p100 (B. afzelii). Thereafter, 30 seronegative healthy controls were tested in LTT-MELISA(R) to determine specificity, 68 patients were tested in parallel to determine reproducibility, and 54 lymphocyte-reactive symptomatic patients were tested before and after antibiotic therapy to assess clinical relevance. Most (86.2%) of the 36.9% (90/244) LTT-MELISA positive patients were seropositive and showed symptoms of active LB. Specificity was 96.7% and reproducibility 92.6%. After therapy, most patients (90.7%) showed negative or markedly reduced lymphocyte reactivity correlating with clinical improvement. This novel LTT-MELISA assay appears to correlate with active LB and may have diagnostic relevance in confirming LB in clinically and serologically ambiguous cases.

Adult↗

PubMed2005

Immunological impact of metals in harbor seals (Phoca vitulina) of the North Sea.

Environmental pollutants may affect the immune system of marine mammals in many areas of the industrialized world. This study provides the first evidence for metal-induced hypersensitivity in harbor seals and demonstrates a relationship between this immunopathy and the level of metals in blood. The concentrations of 20 essential and nonessential elements were analyzed in the blood of 13 harbor seals from the North Sea. In addition, their T-lymphocyte response to metals in terms of hypersensitivity was investigated using a lymphocyte transformation test (LTT) according to the MELISA (memory lymphocyte immuno-stimulation assay) modification. The results showed metal hypersensitivities in 7 of 11 seals investigated in MELISA (data from two seals could not be assessed), reflecting a positive or possible positive reaction in 13 of 154 total single tests. Four animals responded to one metal and three animals to multiple metals. The sensitizing metals were molybdenum (Mo), titanium (Ti), nickel (Ni), chromium (Cr), aluminum (Al), lead (Pb), and tin (Sn). Furthermore, the seals with a Ni-, Al-,.and Cr-sensibilization showed the highest concentrations of these metals in blood. In 8 of the 13 positive cases, elevated blood metal concentrations correlated with the hypersensitivity reaction. Summarizing, we demonstrate in this first pilot study the potential immunological impact of metals in seals, a topic rarely investigated previously. Our results show the value of a combined biological and effect-monitoring tool to investigate pollution-induced immunopathies in live animals.

Animals↗

PubMed2004

Fully automated, internally controlled quantification of hepatitis B Virus DNA by real-time PCR by use of the MagNA Pure LC and LightCycler instruments.

We report on the development of a fully automated real-time PCR assay for the quantitative detection of hepatitis B virus (HBV) DNA in plasma with EDTA (EDTA plasma). The MagNA Pure LC instrument was used for automated DNA purification and automated preparation of PCR mixtures. Real-time PCR was performed on the LightCycler instrument. An internal amplification control was devised as a PCR competitor and was introduced into the assay at the stage of DNA purification to permit monitoring for sample adequacy. The detection limit of the assay was found to be 200 HBV DNA copies/ml, with a linear dynamic range of 8 orders of magnitude. When samples from the European Union Quality Control Concerted Action HBV Proficiency Panel 1999 were examined, the results were found to be in acceptable agreement with the HBV DNA concentrations of the panel members. In a clinical laboratory evaluation of 123 EDTA plasma samples, a significant correlation was found with the results obtained by the Roche HBV Monitor test on the Cobas Amplicor analyzer within the dynamic range of that system. In conclusion, the newly developed assay has a markedly reduced hands-on time, permits monitoring for sample adequacy, and is suitable for the quantitative detection of HBV DNA in plasma in a routine clinical laboratory.

Automation↗

PubMed2002

Quality control in nucleic acid testing--where do we stand?

Quality control has been playing an increasingly important role in the implementation of nucleic acid amplification techniques (NATs) for clinical diagnosis since the introduction of these methods in the early 1990s. Initial multicenter studies involving hepatitis B virus (HBV), hepatitis C virus (HCV), Mycobacterium tuberculosis, and human immunodeficiency virus type 1 (HIV-1) revealed serious problems in specificity (false-positive rates of ca. 40%) and sensitivity, large variations in quantitative results, and a plethora of units (largely not comparable between assays). The problem areas identified included the need for standardized reagents and common units, contamination control mechanisms, inhibition control mechanisms, genotype-independent detection and quantitation, facilitated nucleic acid isolation procedures, clinically relevant dynamic ranges, and internal run controls. Progress made in each of these areas will be discussed. In addition to the above-mentioned problem areas, the value of external quality control of existing and evolving NATs was recognized. To this end, the European Union Quality Control Concerted Action for Nucleic Acid Amplification in Diagnostic Virology was established in May 1998. During its three-and-a-half years of existence, a total of 14 proficiency panels containing 8-13 well-characterized, simulated clinical samples of various viral loads and genotypes were prepared for herpesviruses (herpes simplex virus, human cytomegalovirus), blood-borne viruses (HBV, HCV, HIV-1), enteroviruses, and Chlamydia trachomatis, distributed to up to 20 different countries, and tested by up to 97 different laboratories. The results show dramatic improvement in specificity (false-positive rates <3% for most panels), presumably due to a generally greater expertise of participating laboratories, more frequent use of enzymatic or mechanical contamination control mechanisms, and increased utilization of standardized reagents (commercial kits). However, considerable problems with sensitivity remain (false-negative rates up to 50%), reflecting the high detection limits of some commercial viral load kits still on the market as well as inadequate standardization of quantitation controls between assay systems. In conclusion, although considerable progress has been made, quality control of NATs in clinical diagnosis remains an ongoing challenge.

Insurance, Health, Reimbursement↗

PubMed2002

External quality assessment program for qualitative and quantitative detection of hepatitis C virus RNA in diagnostic virology.

To assess the performance of laboratories in detecting and quantifying hepatitis C virus (HCV) RNA levels in HCV-infected patients, we distributed two proficiency panels for qualitative and quantitative HCV RNA testing. The panels were designed by the European Union Quality Control Concerted Action, prepared by Boston Biomedica Inc., and distributed in May 1999 (panel 1) and February 2000 (panel 2). Each panel consisted of two negative samples and six positive samples, with HCV RNA target levels from 200 to 500,000 copies/ml. Panel 1 had four samples with at least 50,000 copies/ml, and panel 2 had two samples with at least 50,000 copies/ml. Fifty-seven laboratories submitted 45 qualitative and 35 quantitative data sets on panel 1, and 81 laboratories submitted 75 qualitative and 48 quantitative data sets on panel 2. In both panels, about two-thirds of the qualitative data sets and >90% of the quantitative data sets were obtained with commercial assays. With each panel, two data sets gave one false-positive result, corresponding to false-positivity rates of 1.3% and 0.8% for panel 1 and panel 2, respectively. Samples containing at least 50,000 copies/ml were found positive in 97% and 99% of the cases with panel 1 and panel 2, respectively. In contrast, the positive samples containing < or =5,000 copies/ml were reported positive in only 71% and 77% of the cases with panel 1 and panel 2, respectively. Adequate or better scores on qualitative results (all results correct or only the low-positive samples missed) were obtained in 84% (panel 1) and 80% (panel 2) of the data sets. In the analysis of quantitative results, 60% (panel 1) and 73% (panel 2) of the data sets obtained an adequate or better score (> or =80% of the positive results within the range of the geometric mean +/- 0.5 log(10)). Our results indicate that considerable improvements in molecular detection and quantitation of HCV have been achieved, particularly through the use of commercial assays. However, the lowest detection levels of many assays are still too high, and further standardization is still needed. Finally, this study underlines the importance of proficiency panels for monitoring the quality of diagnostic laboratories.

Branched DNA Signal Amplification Assay↗

PubMedDate not supplied

Validity of MELISA for metal sensitivity testing.

OBJECTIVE: This study was carried out to evaluate the reproducibility, sensitivity, specificity, and reliability of the MELISA Test for detecting metal sensitivity in patients with clinical symptoms of a type IV hypersensitivity to metal. DESIGN: Blood from 250 patients was tested in MELISA against up to 20 different metals in 2 to 3 concentrations. The frequency and distribution of metal reactivities, the sensitivity and specificity of nickel reactivity in patients with and without confirmed or suspected sensitivity to nickel, and the roles of lymphocyte concentration and concentration of inorganic mercury were analyzed. In addition, for reproducibility testing, 196 metal tests were performed in duplicate, and intra- and interassay variations of MELISA results were examined in patients patch-test positive for the relevant metal. RESULTS: Among the 250 patients, reactivity to 0, 1, 2, 3, 4, or > or = 5 metals was 26%, 36%, 15%, 12%, 6%, and 5%, respectively. Reactivity was most frequent to nickel (73%), followed by titanium (42%), cadmium (18%) gold (17%), palladium (13%), lead (11%), beryllium (9%), inorganic mercury (8%), tin (8%), and phenylmercury (6%). All patients (n=15) with confirmed or suspected nickel allergy were positive in MELISA, while patients with no suspicion of nickel allergy were either negative (n=6) or very low positive (n=4) in MELISA. MELISA reactivity is directly dependent on lymphocyte concentration: the higher the lymphocyte concentration per test, the stronger the reactivity. Concentrations of inorganic mercury > 0.5 microg/ml cause non antigen-specific (mitogenic) reactions in a majority of patients. The reproducibility rate was 94% using a cut-off of Stimulation Index > or = 3 or 99% using a cut-off of > or = 5. While the absolute intra- and interassay Stimulation Index values may vary, the qualitative results are highly reproducible. CONCLUSION: The MELISA Test is reproducible, sensitive, specific, and reliable for detecting metal sensitivity in metal-sensitive patients.

Humans↗