Comparison of the New, Rapid, and Fully Automated Kryptor TSH receptor Antibodies Assay (B.R.A.H.M.S.) with the Radioimmunological Assay (B.R.A.H.M.S.).

BACKGROUND: Radioimmunoassays, which are often not automated and time-consuming, are gradually being re-placed in medical laboratories by non-radioactive methods that need to be evaluated. The purpose was to compare the measurement of thyroid-stimulating hormone receptor antibodies (TRAb) by the new Brahms' kit using Kryptor TRACE technology and the Brahms' radioimmunoassay. METHODS: We prospectively collected all samples from patients who received thyroid-stimulating hormone receptor antibodies testing in July 2018 at the University Hospital of Brest. The radioimmunoassay used was the Dynotest TRAK human by BRAHMS Diagnostica (Berlin, Germany). The Kryptor method used the BRAHMS TRAK human Kryptor kit performed with the Kryptor Compact Plus system. RESULTS: The inter-assay coefficient variations for the radioimmunological and Kryptor methods were 11.07% and 8.36%, respectively, with the low level quality control and 8.36% and 4.38%, respectively, with the high level quality control. Forty-four patients were included in the study including thirty-two Graves' disease patients in follow-up. The sensitivity of the radioimmunological method for the detection of Graves' disease was 0.94 and the specificity was 0.73. The sensitivity of the Kryptor method was 0.91 and the specificity was 0.91. A non-proportional systematic bias in favor of higher values of TRAb concentrations with the radioimmunological method was observed: slope of 0.93 (0.74 - 1.07, 95% confidence interval) and an intercept of -0.69 IU/L (-1.58 to -0.30, 95% confidence interval). Compared to the Kryptor method, the radioimmunological method tends to overestimate TRAb concentrations by up to 120%. CONCLUSIONS: The fully automated Brahms Kryptor kit using TRACE technology to measure TRAb reduces sampling time and intra- as well as inter-assay variations. The Kryptor kit underestimates the results of TRAb leading to a lower sensitivity and higher specificity compared to the radioimmunoassay. Thus, the new Brahms Kryptor kit has good laboratory performances but the interpretation of the results must still be performed with caution.

Analytical validation of eight methods of thyroglobulin measurement in fine-needle aspiration washouts.

BACKGROUND: Thyroglobulin (Tg) assay in washout fluids of fine needles, after cervical lymph nodes aspiration, is used for detecting metastases from differentiated thyroid carcinomas. Assay methods are the same as for Tg in serum. However, with non-serum samples, methods require extensive validation to notably check for the absence of matrix effect. This study fits this context. Our objectives were to assess analytic performances, in washout fluid, of eight different Tg assay methods and to compare them to validated data in serum. METHODS: Eleven medical laboratories participated in this study. The matrix tested was phosphate-buffer saline containing 1% bovine serum albumin (PBS-1% BSA). Samples used were dilutions, in this buffer, of Certified Reference Material (CRM 457). We verified, for all methods, the limit of detection, precision, linearity, trueness and accuracy. RESULTS: In PBS-1% BSA, the functional sensitivities (FS) were comparable to those expected for serum. All the methods were linear. The relative biases of trueness were between -24.5 and 10.2% around 1 µg/L. Total analytical error was ≤40% near the functional sensitivity values. CONCLUSION: No quantitatively important matrix effect was observed. All the methods showed their ability to measure Tg in PBS-1% BSA, over the concentration range of interest, with acceptable total analytical error. We validated the functional sensitivity value as a decision threshold in thyroidectomized patients after treatment and with low concentrations of serum Tg.

Correlation of biological parameters with placental parameters and pregnancy outcomes in pre-eclamptic women.

INTRODUCTION: Pre-eclampsia is characterized by maternal vascular malperfusion and chronic inflammation in placenta. Our purpose was to investigate the potential correlation of biological parameters with placental parameters and pregnancy outcomes in pre-eclamptic women. METHODS: Pre-eclamptic women were identified by interrogation of the Medical Registry Department in six French maternities between April 2013 and June 2018. Histological parameters in placentas (weight, macroscopic and microscopic lesions), baseline maternal characteristics and pregnancy outcomes (course of pregnancy, newborns' characteristics) were collected. Biological parameters were blood cell ratios (Platelet-to-Lymphocyte Ratio (PLR), Neutrophil-to-Lymphocyte Ratio (NLR)) collected at delivery and Placental growth factor (PlGF) measured in women with an available first trimester serum sample. Correlations of blood cell ratios and PlGF levels with placental parameters and pregnancy outcomes were assessed by Pearson's correlation test for quantitative parameters and by logistic regression analysis for qualitative parameters. RESULTS: 202 pregnancies were included, among which 68 had a first trimester PlGF quantification. No correlation was found between biological parameters and placental lesions. Low PLR was correlated with low placental weight (r = 0.156, p = 0.036) and with low birth weight (r = 0.179, p = 0.015). Low PlGF was correlated with long time from pre-eclampsia diagnosis to delivery (r = -0.250, p = 0.048). CONCLUSIONS: There is no correlation between biological parameters and placental lesions in pre-eclamptic women. Yet, low PLR at delivery is correlated with low placental and birth weights. Moreover, low first trimester PlGF is correlated with long time from pre-eclampsia diagnosis to delivery.

First trimester serum biomarkers in pregnancies complicated with placental chronic inflammation.

OBJECTIVE: This study aimed at determining if first trimester serum biomarkers could predict adverse pregnancy outcomes associated with villitis (VUE) and chronic intervillositis of unknown etiology (CIUE). STUDY DESIGN: Between January 2013 and June 2018, we selected from pathology department files placentas with VUE or CIUE associated with VUE and control placentas with available first trimester Down syndrome screening results. First trimester PAPP-A and ßhCG levels were recorded. Placental growth factor (PlGF) levels were measured in patients with an available first trimester serum sample. Histological findings in placentas, course of pregnancies and newborns' characteristics were compared between cases and controls. RESULTS: 78 cases and 75 controls were included. In cases, there were 21,8% intrauterine growth restriction (IUGR), 30,8% small for gestational age (SGA). Compared to controls, placentas from cases were smaller (425 g [IQR 370-480] vs 460 g [IQR 390-523], p = 0,03), showed more maternal vascular malperfusion features (79,5% vs 22,7%, p < 0,0001) and more fetal vascular malperfusion features (33,3% vs 12%, p = 0,002). Cases had lower PlGF (29,74 pg/ml [IQR 19,74-36,17] vs 36,37 pg/ml [IQR 27,36-49,13], p = 0,007) and ßhCG levels (0,74 MoM [IQR 0,53-1,12] vs 1,00 MoM [IQR 0,72-1,53], p = 0,002) than controls. These differences resulted from lower PlGF levels in VUE patients compared to CIUE associated with VUE patients and controls (28,35 vs 34,05 and 36,37 pg/ml, p = 0,01) and from lower ßhCG levels in CIUE associated with VUE patients compared to VUE patients and controls (0,65 vs 0,86 and 1, p = 0,005). CONCLUSION: Low first trimester PlGF levels in cases, especially in VUE patients, suggest that reduced angiogenesis is involved in adverse pregnancy outcomes related to VUE.

Preanalytical stability of maternal serum markers hCGß and PAPP-A.

Down syndrome maternal serum marker screening is based on a risk calculation including the free ß - human chorionic gonadotropin (hCGß) and pregnancy-associated placenta protein type A (PAPP-A). The aim of this study was to define the pre-analytical conditions of stability of these markers both in whole blood at 15-25 ÌŠC and, after centrifugation, in serum at 4-8 ÌŠC. 158 patients were included in the study. Two automated workstations were used for assays, Cobas 8000e602, Roche Diagnostics (58 patients tested) and DELFIAXpress, PerkinElmer (100 patients tested). The stability of markers was studied in whole blood (15-25 ÌŠC) 2, 4, 6 and 8 hours after sampling and in serum stored after centrifugation at 4-8 ÌŠC at 24, 72 and 120 hours. Variations were defined by (CT - C2)/C2, C2 being the marker concentration at 2 hours and CT the concentration at time T. In whole blood kept for 8 hours at 15-25 ÌŠC, hCGß increased by a mean 2.4%, whereas the mean increase of PAPP-A was < 1%. In the serum kept for 5 days at 4-8̊C, the mean increase of hCGß was 4.2%, with no change in PAPP-A. The impact of these variations on risk calculation is low. In conclusion, maternal serum can be store 8 hours at 15-25̊C in whole blood and 5 days at 4-8̊C after centrifugation and serum separation for Down syndrome maternal serum screening.

Thyroglobulin assay in fluids from lymph node fine needle-aspiration washout: influence of pre-analytical conditions.

The aim of this study was to evaluate the pre-analytical factors contributing to uncertainty in thyroglobulin measurement in fluids from fine-needle aspiration (FNA) washout of cervical lymph nodes. We studied pre-analytical stability, in different conditions, of 41 samples prepared with concentrated solutions of thyroglobulin (FNA washout or certified standard) diluted in physiological saline solution or buffer containing 6% albumin. In this buffer, over time, no changes in thyroglobulin concentrations were observed in all storage conditions tested. In albumin free saline solution, thyroglobulin recovery rates depended on initial sample concentrations and on modalities of their conservation (in conventional storage tubes, recovery mean was 56% after 3 hours-storage at room temperature and 19% after 24 hours-storage for concentrations ranged from 2 to 183 µg/L; recovery was 95%, after 3 hours or 24 hours-storage at room temperature, for a concentration of 5,656 µg/L). We show here that these results are due to non-specific adsorption of thyroglobulin in storage tubes, which depends on sample protein concentrations. We also show that possible contamination of fluids from FNA washout by plasma proteins do not always adequately prevent this adsorption. In conclusion, non-specific adsorption in storage tubes strongly contributes to uncertainty in thyroglobulin measurement in physiological saline solution. It is therefore recommended, for FNA washout, to use a buffer containing proteins provided by the laboratory.

Development of medians generators for the calculation of MoM for the first-trimester Down syndrome maternal serum markers.

The Down syndrome risk calculation software, Fast Screen pre I plus, from Thermo Fisher Scientific, converts the First-trimester maternal serum markers concentrations (PAPP-A and hCGß) into degrees of extreme (DoE). To meet the requirements of the French legislation as well as those of the certified biologists for Down syndrome screening, a conversion tool for DoE into multiple of the median (MoM), intermediate reference value for the screening, was developed. In absence of any median polynomial allowing to obtain MoM easily, the calculations were made first on the basis of the polynomials centiles from the software. The subsequent reviews of the risk algorithms showed the fragility of this approach based on the Gaussian hypothesis. Medians generators, calculated from the data of the Kryptor club's biologists, were established.

Serum levels of 25(OH)D are not associated with venous thromboembolism in the elderly population. A case-control study.

The prevalence of both vitamin D deficiency and venous thromboembolism (VTE) is important in the elderly. Previous studies have provided evidence for a possible association between vitamin D status and the risk of VTE. Thus, we aimed to investigate the association between vitamin D levels and VTE in the population aged 75 and over included in the EDITH case-control study. The association between vitamin D status and VTE was analysed. We also analysed the monthly and seasonal variations of VTE and vitamin D. Between May 2000 and December 2009, 340 elderly patients (mean age 81.5 years, 32% men) with unprovoked VTE and their controls were included. The univariate and multivariate analysis found no significant association between serum levels of vitamin D and the risk of unprovoked VTE. In the unadjusted analysis, a higher BMI was statistically associated with an increased risk of VTE (OR 1.09; 95% CI 1.05-1.13) whereas a better walking capacity and living at home were associated with a decreased rate of VTE: OR 0.57; 95% CI 0.36-0.90 and 0.40; 95% CI 0.25-0.66, respectively. Although not significant, more VTE events occurred during winter (p=0.09). No seasonal variations of vitamin D levels were found (p=0.11). In conclusion, in contrast with previous reports our findings suggest that vitamin D is not associated with VTE in the elderly population.

Interaction of low dose of fish oil and glucocorticoids on insulin sensitivity and lipolysis in healthy humans: A randomized controlled study.

SCOPE: This study examined the interaction of fish oil (FO) with dexamethasone on glucose and lipid metabolisms in healthy subjects. METHODS AND RESULTS: The study included two consecutive parts. Part A (randomized) in 16 subjects studied the effects of dexamethasone (2 days, 2 mg/day) versus placebo (lactose), part B (two parallel subgroups of eight) studied the interaction of FO (3 wk, 840 mg/day of EPA + DHA) with dexamethasone. Insulin sensitivity of lipolysis (d5-glycerol infusion + microdialysis), endogenous glucose production, and muscle glucose uptake were assessed by a three-step hot insulin clamp and substrate oxidation by indirect calorimetry. Dexamethasone induced liver and peripheral insulin resistance, an increase in fat oxidation, and a decrease in suppression of plasma nonesterified fatty acids (NEFAs). FO amplified the effects of dexamethasone by increasing liver and muscle insulin resistance, by reducing suppression of plasma NEFAs and fat oxidation and by increasing adipose tissue (AT) lipolysis. CONCLUSION: FO, given at a moderate dose in healthy subjects prior to a very short-term (2 days) low dose of a synthetic glucocorticoid, worsened its deleterious effects on insulin sensitivity. The enhancing effect of FO on fat oxidation and AT lipolysis might be a protective effect toward an increase in fat mass.

[Pre-analytical stability before centrifugation of 7 biochemical analytes in whole blood]. / Impact sur le dosage en sang total de sept analytes biochimiques de la durée de conservation avant centrifugation des échantillons.

The pre-analytical stability of 7 biochemical parameters (parathyroid hormone -PTH-, vitamins A, C E and D, 1,25-dihydroxyvitamin D and insulin) at +4 °C, was studied on whole blood samples before centrifugation. The impact of freezing at -20°C was also analyzed/performed for PTH and vitamin D. The differences in the results of assays for whole blood samples, being kept for different times between sampling time and analysis, from 9 healthy adults, were compaired by using a Student t test. The 7 analytes investigated remained stable up to 4 hours at +4°C in whole blood. This study showed that it is possible to accept uncentrifuged whole blood specimens kept at +4°C before analysis. PTH is affected by freezing whereas vitamin D is not.

Effects of oral and transdermal 17 beta-estradiol combined with progesterone on homocysteine metabolism in postmenopausal women: a randomised placebo-controlled trial.

Mild hyperhomocysteinemia is a risk factor for both ischaemic heart disease and venous thromboembolism. The effects of transdermal estrogen replacement therapy (ERT) on homocysteine metabolism in postmenopausal women have scarcely been investigated. This clinical trial aimed to estimate the effects of combined hormone replacement therapy on the fasting total homocysteine levels according to the estrogen route of administration. We enrolled 196 postmenopausal women, who were randomly allocated to receive on a continuous basis either 1mg of 17 beta-estradiol orally (n = 63) or 50 microg transdermally (n = 68) per day, both combined with a daily intake of 100 mg progesterone, or placebo (n = 65) over a period of 6 months. Neither oral nor transdermal ERT significantly affected total plasma homocysteine levels or red-blood cell folate levels. However, oral ERT significantly decreased plasma vitamin B12 levels compared to placebo (mean relative variation difference over 6 months between oral ERT and placebo: -11.7% (95%CI, -21 to -2%) whereas transdermal ERT did not display any significant effects. Our data show that transdermal ERT as well as low dose of oral ERT does not significantly affect the homocysteine metabolism. This finding does not support a role for transdermal estrogen in the prevention of ischaemic heart disease in postmenopausal women.

[Comparative study of parathormone and vitamin D measurements by three automats: ADVIA Centaur XP® (Siemens), ISYS® (IDS) and Liaison® (Diasorin)]. / Étude comparative des dosages de parathormone et de vitamine D sur 3 automates : Advia Centaur XP (®) (Siemens), Isys(®) (IDS) et Liaison(®) (Diasorin).

Vitamin D (Vit.D) and parathormone (PTH) measurements are usually prescribed for phosphocalcic metabolism assessment and, especially for Vit.D, more and more frequently for other pathologies. In order to step up to automated techniques for these analysis in our laboratory, we tested 3 devices: ADVIA Centaur XP(®) (Siemens), ISYS(®) (IDS) and Liaison(®) (Diasorin), which allow to simultaneously quantify Vit.D and PTH. The aim of this study was to evaluate the fidelity of these methods as well as study the correlation between them and the radioimmunological techniques previously used in our laboratory : « ELSA PTH ¼ (Iba Cisbio International) and « 25-OH D ¼ (IDS). The comparison of PTH analysis was performed on a population of chronic renal failure patients undergoing haemodialysis. According to our study, the 3 devices show acceptable analytical performances; anyway the measurements realized on the ISYS analyzer are the ones showing the best results in terms of fidelity, and the closest results to those obtained with the RIA reference techniques.

The hook effect in calcitonin immunoradiometric assay: a case report.

UNLABELLED: The hook effect, which has long been detected and documented for immunoradiometric assays (IRMA) such as those measuring prolactin or thyroglobulin, occurs when the serum antigen level is extremely high, thus inducing a bias in the methodology of measurement. RESULTS: We report the case of an 80-year-old man with confirmed medullary thyroid carcinoma (MTC). In the case reported here, the clinical status of the patient contrasts with his tumor antigen, serum calcitonin (CT), concentrations. The measured increased CT concentrations revealed the presence of a hook effect. This phenomenon occurs due to an excess of antigen during the one-step IRMA where the signal antibodies, bound to the non-captured antigens, are washed out during the measurement, inducing the loss of signal. Aiming to prevent the "hook effect", successive dilutions of the same sample of serum were done. CONCLUSIONS: Previous studies have shown when one-step IRMA reveals high concentrations of a tumor serum antigen (i.e. prolactin or thyroglobulin), a two-step IRMA or a systematic 1:10 dilution of the serum sample prevents the formation of the "hook effect". In our case report, the CT "hook effect" formation was prevented by performing serial dilutions of the serum sample.

Targeted screening for prostate cancer in high risk families: early onset is a significant risk factor for disease in first degree relatives.

PURPOSE: Targeted screening for prostate cancer in high risk families is generally suggested by ages 40 to 45 years in first degree relatives. We support this concept by reporting higher risk and earlier onset of the disease in these families. MATERIALS AND METHODS: We proposed serum prostate specific antigen (PSA) testing in 40 to 70-year-old first degree relatives of 435 patients with prostate cancer treated between July 1994 and June 1997. A previous systematic genealogical analysis allowed us to define the familial prostate cancer status of each patient as sporadic or familial. RESULTS: Of the 747 potential candidates 442 (59%) accepted into the study have been screened, including 240 who were 40 to 49 years old (mean age 44.8) and 202 who were 50 to 70 years old (mean age 57.4). Two of the 240 subjects (0.8%) had PSA greater than 4 ng./ml. in the 40 to 49-year-old group. Prostate biopsies were negative in 1 relative but diagnostic for prostate cancer in the other. In the 50 to 70-year-old group 25 of 202 subjects (12.4%) had a PSA of greater than 4 ng./ml. Prostate cancer was diagnosed in 9 individuals (4.5%), 9 had negative biopsy results, 1 died before biopsy and 6 refused biopsy. The proportion of relatives with PSA greater than 4 ng./ml. and prostate cancer detection was not different according to familial status (sporadic or familial) but it was significantly higher in first degree relatives with early onset prostate cancer in the family at ages younger than 65 years (p = 0.037 and 0.012, respectively). CONCLUSIONS: Our results emphasize the usefulness of PSA screening in high risk families, including those without obvious hereditary features. Furthermore, early onset prostate cancer is a significant risk factor for prostate cancer in first degree relatives.