Anne-Sophie Gauchez

Variability among TSH Measurements Can Be Reduced by Combining a Glycoengineered Calibrator to Epitope-Defined Immunoassays

Objectives: Measuring protein markers with variable glycosylation, such as thyroid-stimulating hormone (TSH), with high accuracy is not an easy task. Despite highly sensitive third-generation tests, discrepancies among TSH assays still remain unsolved and are the focus of important standardization efforts. Earlier work from our group showed that a lack of similarity in epitope expression between standards and samples may account for discordant hormone measurements. In this study, we aimed at producing a glycoengineered TSH with serum-type glycosylation and compared its immunological behavior to that of the international standards. Study Design: Recombinant glycoengineered TSH (rgTSH) was produced in glycoengineered Chinese hamster ovary cells to express a highly sialylated TSH and tested in newly designed assays. Two groups of assays targeting defined epitopes were constructed and TSH levels were estimated in a panel of 84 clinical samples (2.1-22.4 mIU/l) based on the use of the current 3rd IS 81/565, the 1st IRP 94/674 and rgTSH calibrations. Results: Calibration based on rgTSH was found to significantly reduce the percentage difference means of assays compared to the pituitary standard. We also found that a switch from a mIU/l (3rd IS 81/565) to ng/l (rgTSH) basis can be established within the normal as well as in the mid to upper normal range of TSH levels. Of interest, TSH assays targeting the main immunogenic region displayed variable TSH values, indicating that, in this region, epitopes should be defined for assays to deliver similar values. Conclusions: A glycoengineered TSH with serum-type glycosylation proved to be a new calibrator efficient in harmonizing TSH values.

Evaluation of a human anti-mouse antibody rapid test for patients requiring radio-immunodiagnostic

Monoclonal antibodies (mAbs) play a significant part in the diagnostic and therapeutic arsenal. They are used in many fields of medicine, ranging from in vitro diagnosis to the treatment of multiple pathologies in such diverse areas as infectious diseases, immunology and oncology. The first mention of the use of radiolabeled antibodies dates from the 1950s. However, from the first tests, several problems with the use of radiolabeled antibodies appeared. One of these involved the use itself of the antibodies because they caused an immune response and the production of human anti-mouse antibodies (HAMAs) [1]. Complications related to this immunization are very rare and have a high inter-individual variability [2–4]. It is widely accepted that the presence of these heterophilic antibodies could influence the effectiveness of the immunotherapy and immunoscintigraphy, and could also be considered as responsible for analytical interference in immunoassays [5, 6]. Indeed, HAMAs bind to the newly administered mAbs, leading to the formation of immune complexes (HAMA-mAb) that may decrease the therapeutic efficacy and diagnostic value of the new mAb conjugates. Similarly, allergic reactions could occur, motivating the search for HAMAs before any reintroduction of mAbs [7]. Current tests for HAMA detection do not allow one to quickly obtain the status of patients. To develop a simple, rapid and reliable test for monitoring the presence of HAMA in some patients, Milenia GmbH (Hamburg, Germany) has recently proposed a new qualitative test based on the principle of a rapid lateral flow test on a strip. We proposed to evaluate this Quicktest on our cohort of patients who had tested positive for HAMAs using the quantitative Medac HAMA-ELISA test. Serum samples from patients positive for HAMA were obtained from a serum bank collected between 1992 and 1999 in the Nuclear Medicine Department of Grenoble University Hospital. The patients had received radioimmunotherapy and subsequently developed HAMAs demonstrated using the quantitative Medac test. Sera were estimated again in 2014 using the same test to verify the persistence of expression of HAMA. This test is a one-step enzyme immunoassay for the quantitative determination of HAMA in serum realized in 1 h. The test is calibrated against anti-mouse IgG antibodies. The measuring range is from 40 to 2000 ng/mL. Samples and peroxidase-labeled mouse IgG conjugate are added together to the plate that is pre-coated with mouse IgG (antigen). The HAMAs bind to the solid phase and to peroxidase-labeled mouse IgG. Then, there is an incubation step with a tetra-methyl benzidine substrate. The reaction is stopped by the addition of sulfuric acid, and the absorption is read photometrically at 450 nm. According to the manufacturer, the limit *Corresponding author: Marie-Dominique Desruet, PhD, PharmD, Radiopharmacy/ Nuclear Medicine Department, Grenoble University Hospital, CS 10217 38043 GRENOBLE cedex 9, France, Phone: + (0)33 476 769 410, Fax: + (0)33 476 767 108, E-mail: mddesruet@chu-grenoble.fr; and Pharmacy Department, Grenoble University Hospital, Grenoble, France Chloé Lamesa: Pharmacy Department, Grenoble University Hospital, Grenoble, France; and UMR-S INSERM 1039 Bioclinical Radiopharmaceutical Unit, Grenoble, France Anne-Sophie Gauchez: UMR-S INSERM 1039 Bioclinical Radiopharmaceutical Unit, Grenoble, France; and Radioactivity Platform, Biology Department, Grenoble University Hospital, Grenoble, France Roseline Mazet and Luc Foroni: Pharmacy Department, Grenoble University Hospital, Grenoble, France Daniel Fagret: UMR-S INSERM 1039 Bioclinical Radiopharmaceutical Unit, Grenoble, France; and Nuclear Medicine Unit, Imaging Department, Grenoble University Hospital, Grenoble, France

Subclinical Hypothyroidism: is it Really Subclinical with Aging?

No recent study has focused on clinical features of subclinical hypothyroidism (SCH), especially in older patients. TSH measurement has remarkably evolved these last 20 years and thus reconsideration is needed. In our prospective multicenter study (2012-2014) including 807 subjects aged <60 years (<60y) and 531 subjects ≥60 years (≥60y), we have monitored 11 hypothyroidism-related clinical signs (hCS) together with TSH, FT4, FT3 and anti-thyroperoxidase antibodies values. hCS expression has been compared in patients with SCH vs euthyroidism in each age group. The number of hCS above 60y of age were found to be more elevated in the euthyroid population (1.9 vs 1.6, p<0.01) than in the SCH population (2.3 vs 2.6, p=0.41) while increase in hCS is limited to SCH subjects in the <60y group (p<0.01). The percentage of subjects with at least 3 signs increased with SCH in the <60y group (42.6% vs 25.0%, p<0.01) but not ≥60y (34.4% vs 33.9%, p=0.96). In older individuals, only three hCS could be related to both SCH and a decreased T3/T4-ratio (0.26 vs 0.27, p<0.01), suggesting either a reduced activity of TSH, or an adaptive response with aging. While hCS are clearly associated with SCH in patients <60y, they are not so informative in older subjects. TSH measurements carried out on the basis of hCS need to be interpreted with caution in aged patients. A reassessment of the TSH reference range in older patients is clearly needed and should be associated to more appropriate monitoring of thyroid dysfunction

Fetal hypothyroidism induced by maternal anti-TSH receptor blocking antibodies and complicated by polyhydramnios despite the absence of goiter. Treatment by intra-amniotic injections of levothyroxine

Fetal hypothyroidism induced by maternal anti-TSH receptor blocking antibodies and complicated by polyhydramnios despite the absence of goiter. Treatment by intra-amniotic injections of levothyroxine

Diagnosis, Clinical Work Up, TNM Classification, Markers

Prostate cancer (PCa) is the most common malignancy in developed countries, and the incidence is rapidly rising. Today, early detection is mostly induced by a large prescription of prostate-specific antigen (PSA) blood test. Although PSA remains the gold standard for diagnosis, clinical rectal examination is essential for staging prostate cancer and deciding treatment.