Fluorometric Enzyme Immunoassay Research Articles

Comparative analysis of ELISA, FEIA and CLIA techniques in diagnostic immunoassays for thyroid stimulating hormone: A cross-sectional study form Morang, Nepal

Background: TSH and circulating thyroid hormones are measured in serum as part of a thyroid function test (TFT), which evaluates the thyroid gland's ability to produce and regulate thyroid hormones. The objective of the current study was to compare the technical performance between ELISA, FEIA and the currently marketed automated CLIA for measuring the TSH level.
 Material and methods: A total of 1,200 participants were enrolled in the study. Subjects were chosen randomly, either OPD patients or inpatients of Birat Medical College Teaching Hospital. Chemiluminescence Immunoassay (CLIA), Enzyme-linked Immunoassay (ELISA) and Fluorometric Enzyme Immunoassay (FEIA) technologies were performed.
 Results: The total number of patients (1200) was categorized into group I, normal TSH (n = 680); group II, elevated TSH (n = 300); and group III, decreased TSH (n = 220). CLIA and FEIA based results were significantly more sensitive compared to ELISA results for TSH detection.
 Conclusion: Chemiluminescence assay system, when compared to FEIA and ELISA, is far superior in terms of accuracy and sensitivity for reporting both subnormal and above normal TSH levels and can be helpful in the detection of subclinical thyroid dysfunction (SCTD) and in the screening of thyroid diseases.

Association of C-peptide level with peripheral neuropathy in type 2 diabetes: An observational cross-sectional preliminary study

Backround and aimsPeripheral neuropathy is the most common microvascular complication of diabetes mellitus. In subjects with type 1 diabetes (T1D) relationship of C-peptide levels and neuropathy has been observed in several studies, however, there are very few studies in type 2 diabetes (T2D) subjects. In this study we aim to assess the association of C-peptide levels with peripheral neuropathy in Indian subjects with T2D. MethodOne hundred patients of T2D were included in this study. Clinical and laboratory parameter was assessed for all participants. The C-peptide level was measured by fluorometric enzyme immunoassay method. Assessment of diabetic peripheral neuropathy was based on diabetic neuropathy symptom score and the diabetic neuropathy examination scores. ResultsTotal 100 patients completed the study. Mean age of subjects was 60.03 years and male: female ratio was 1.17. Peripheral neuropathy was detected in 47% of subjects evaluated. Subjects were further divided in to neuropathy group and no-neuropathy group for analysis. Age in neuropathy group was significantly higher than no-neuropathy group [65.62 ± 10.5 vs 55.08 ± 9.41 yrs (p-value <0.0001)] and similarly duration of T2D was significantly higher in neuropathy group [10.11 ± 6.13 vs 4.16 ± 3.7 yrs (p-value <0.0001)]. Importantly mean fasting C-peptide (2.27 ± 0.98 vs 3.12 ± 0.84 ng/ml) and mean post meal C-peptide (4.27 ± 1.34 vs 5.33 ± 0.89 ng/ml) were significantly lower in neuropathy group compared to no-neuropathy group. An association of HbA1c level and neuropathy was statistically not significant (p = 0.793). ConclusionSerum C-peptide concentrations are associated with peripheral neuropathy in T2DM patients, independent of the degree of glycemic control.

Mechanisms for inhibitory effect of ALDH2 on doxorubicin-induced cytotoxicity in C2C12 myogenic cell line

To investigate the mechanisms for inhibitory effect of aldehyde dehydrogenase 2 (ALDH2) on doxorubicin (DOX)-induced cytotoxicity in C2C12 myogenic cell line. Cell apoptosis was evaluated by flow cytometry and the activity of capase-3/7. The relative content of reactive oxygen species (ROS) and 4-hydroxynonenal (4-HNE) were detected by chemical fluorometric enzyme immunoassay. The protein and mRNA expression of ALDH2, Bcl-2, NADPH oxidase 2 (NOX2) and the cytoplasmic subunit p-p47PHOX were evaluated by Western blot and quantitative PCR, respectively. Overexpression of ALDH2 attenuated DOX-induced cell toxicity (increase in apoptosis and inhibition of proliferation), which were reversed by downregulation of ALDH2. Overexpression of ALDH2 reduced p47PHOX phosphorylation levels, and suppressed activation of NOX2 and ROS production, which were reversed by downregulation of ALDH2. Moreover, apocynin, an inhibitor of NOX, reduced the cytotoxicity of DOX concomitantly with a decrease in phosphorylation of p47PHOX, ROS production and caspase-3/7 activity, and an increase in the activity and expression of ALDH2. DOX-induced cytotoxicity is related to increase of intracellular oxidative stress, which is involved in unregulation of NOX2 and downregulation of ALDH2. Activation of ALDH2 could exert cytoprotection via inhibiting NOX2-dependent ROS production.

Structural modifications of the prostate in hypoxia, oxidative stress, and chronic ischemia.

PurposeClinical studies have reported a correlation between pelvic ischemia and voiding dysfunction in elderly men. The aim of this study was to identify and compare prostate structural modifications in cultured cells and in a rabbit model after exposure to hypoxia, oxidative stress, and chronic ischemia.Materials and MethodsCultured human prostate smooth muscle cells (SMCs), epithelial cells (ECs), and stromal cells (SCs) were incubated under normoxia, hypoxia, and oxidative stress conditions by use of a computerized oxycycler system. We developed a rabbit model of chronic prostate ischemia by creating aorto-iliac arterial atherosclerosis. Markers of oxidative stress were examined by using fluorometric analysis and enzyme immunoassay. Prostate structure was examined by using Masson's trichrome staining and transmission electron microscopy (TEM).ResultsLipid peroxidation was found in SMCs exposed to hypoxia and in all cell types exposed to oxidative stress. We identified protein oxidation in ECs exposed to hypoxia and in all cell types exposed to oxidative stress. Markers indicating oxidative damage were present in chronically ischemic rabbit prostate tissue. These reactions were associated with DNA damage. Prostate ischemia resulted in epithelial atrophy, loss of smooth muscle, and diffuse fibrosis. TEM showed swollen mitochondria with degraded cristae, loss of membrane, loss of Golgi bodies, degenerated nerves, and disrupted cell-to-cell junctions.ConclusionsHuman prostate cells exhibited differential reactions to hypoxia and oxidative stress with widespread DNA damage. Structural modifications in ischemic prostate tissue were similar to those in cells exposed to oxidative stress. Structural changes due to ischemia and oxidative stress may contribute to prostatic noncompliance in aging men.

Comparison of measurement of serum tsh by two 3rd generation techniques.

The present study compared the measurement of serum TSH by two 3 rd generation methods based on different principles i.e. Chemiluminesence (CLIA) and Fluorometric enzyme immuno-assay (FEIA). Main aim of the study was to evaluate the usefulness of the more sensitive assay method in patients with thyroid disease and non-thyroid illness (NTIs). Blood samples were withdrawn randomly from 125 subjects attending OPD and same sample was analyzed for TSH on two equipment’s based on methods with different principles.i.e. CLIA (Chemiluminesence) and FEIA (AIA-360). Out of 125 serum samples, the levels of TSH were within normal range in 68 subjects, were above normal range in 35 subjects and were below normal range in 22 subjects by both the methods. A specific value for TSH could not be reported by FEIA based equipment-AIA-360 in 14 (63.6%) out of total 22 hyperthyroid subjects taken up for the study. The TSH values in 35 hypothyroid subjects obtained by two different method was also significantly (p< 0.0001) different. This clearly indicates that the Chemiluminesence assay system is far superior with respect to precision and sensitivity as compared to FEIA for reporting subnormal TSH levels as well as above normal TSH and can be useful in detection of SCTDs (subclinical thyroid dysfunction) and in screening of thyroid diseases.

117 SERUM PROGESTERONE CONCENTRATION AND EMBRYO DIAMETER ON DAY 7 POST-OVULATION IN MARES

Monitoring serum progesterone (P4) concentrations has been a routine practice for determining phases of the oestrous cycle in mares. The use of maternal serum P4 concentration to estimate embryo size would be beneficial in determining the proper collection time for equine embryos destined for cryopreservation and transfer. Therefore, the objective of this study was to determine if embryo diameter was correlated with maternal serum P4 concentration on Day 7 post-ovulation. Light horse mares of various breeds (n = 40) were used in this study. The mares were in good body condition and ranged in age from 3 to 18 yrs. Oestrus detection was performed first with a stallion followed by reproductive tract examinations via transrectal ultrasonography to predict ovulation. Blood samples were collected via jugular venipuncture before artificial insemination (AI) (Day 0) and again on Day 7 post-ovulation. Blood samples were allowed to clot, then centrifuged for 10 min at 2600 × g and serum stored at –20°C until assayed. Each donor mare was inseminated with extended fresh semen every other day until ovulation. On Day 7 post-ovulation, a nonsurgical embryo collection was performed on inseminated mares by a single technician using a standard equine uterine flushing procedure. Only blood samples taken from mares in which an embryo was collected were analysed (n = 18). Blood samples were collected from similar noninseminated mares (n = 12) from the same research herd on the day of expected AI (Day 0) and again on Day 7 post-ovulation. Serum progesterone was determined using a P4 fluorometric enzyme immunoassay (FEIA; Tosoh Bioscience, Inc., South San Francisco, CA). Embryo diameter was measured including the zona pellucida using Micron Software (Westover Scientific). Mean P4 concentrations (±s.e.) for all inseminated donors producing an embryo were 0.49 ± 0.1 ng mL–1 at Day 0 and 12.9 ± 1.2 ng mL–1 on Day 7 post-ovulation. Mean P4 concentration of donors on Day 7 post-ovulation with embryo diameters of &lt;400 μm (n = 7) was not different from donors producing embryos &gt;400 μm (n = 10) and were 11.2 ± 1.7 ng mL–1 and 14.1 ± 1.7 ng mL–1, respectively. One mare produced viable twin embryos (284 and 373 μm in diameter) and had a P4 level of 35 ng mL–1 on Day 7 post-ovulation, and was not included in the data analysis. Mean P4 concentrations in the noninseminated mares were 0 0.68 ± 0.2 ng mL–1 on Day 0 and 12.3 ± 1.6 ng mL–1 on Day 7 post-ovulation and were not different from embryo-producing mares regardless of embryo size. From this preliminary study, that there is no evidence that embryo diameter is correlated with maternal progesterone concentrations on Day 7 post-ovulation.

Use of commercial anti–penicillin IgE fluorometric enzyme immunoassays to diagnose penicillin allergy

The intermittent unavailability of penicilloyl-polylysine since September 2000 has focused interest on commercial anti-penicillin IgE fluorometric enzyme immunoassay (FEIA) tests to evaluate penicillin allergy. There has been no published comparison of commercial anti-penicillin IgE FEIAs and penicillin skin testing performed in the United States. To determine whether the current commercial anti-penicillin IgE FEIAs can replace or augment penicillin skin testing and oral challenges when evaluating individuals with a history of penicillin allergy for future therapeutic penicillin tolerance. A prospective convenience sample of 150 individuals with a history of penicillin allergy were evaluated between January 23, 2007, and August 4, 2009, with both penicillin skin tests and commercial anti-penicillin IgE FEIAs to penicillin G, penicillin V, and amoxicillin. All individuals with a negative penicillin skin test result underwent oral penicillin class antibiotic challenges. All individuals with a positive anti-penicillin IgE FEIA result also underwent oral penicillin class antibiotic challenges. Six individuals (4.0%; 95% confidence interval [CI], 0.9% to 7.1%) had positive penicillin skin test results, and none had positive FEIA results. Four individuals (2.7%; 95% CI, 0.1% to 5.3%) had positive FEIA results, and none had positive penicillin skin test results. Three individuals (2.0%; 95% CI, -0.2% to 4.2%) had positive oral challenge results, 1 with hives at 6 hours after challenge and 2 with delayed-onset (at >24 hours) nonurticarial rashes, and none had positive FEIA results. The current commercial anti-penicillin IgE FEIAs are not useful in diagnosing penicillin allergy in patients with remote histories of penicillin allergy. Penicillin skin testing and, if the results are negative, an oral challenge remain the criterion standard tests to determine therapeutic penicillin tolerance.

New fluorometric enzyme immunoassay for 17β-estradiol by homogeneous reaction using biotinylated estradiol

A new fluorometric enzyme immunoassay for 17β-estradiol (E2) using biotinylated estradiol (BE) as a probe ligand, is described. In this method, E2 is detected indirectly by a solid-phase avidin–biotin binding assay, in which the biotin is immobilized on a microtiter plate (biotin-plate). After the competitive reaction between E2 and BE for the anti-E2 antibody in solution, the free E2 and BE are separated from the bound forms by means of ultrafiltration. The concentration of BE in the solution is determined from the reaction between the biotin immobilized on the plate and the free BE for the limited biotin binding sites of avidin conjugated with horseradish peroxidase (avidin–HRP), which is added to the solution. The enzymatic reaction of HRP was measured by a fluorometric analysis with the QuantaBlu™ Fluorogenic Peroxidase Substrate (QFPS) in order to detect of the avidin–biotin binding with a high degree of sensitivity. The detection limit and linear range for the determination of E2 were 0.12 nM and from 0.12 to 25 nM, respectively. The relative standard deviations (R.S.D.) for the E2 assay were between 2.2 and 9.1% ( n = 3). The cross-reactivity for several other estrogens was also evaluated.

EVALUATION OF THE STRATUS® CS FLUOROMETER FOR THE DETERMINATION OF PLASMA MYOGLOBIN

Circulating myoglobin is recognized as an early and sensitive marker of acute coronary diseases. Long turnaround time of myoglobin assays jeopardize their clinical utility. We evaluated the analytical performance of the Stratus® CS fluorometric enzyme immunoassay based on dendrimer technology, and claimed to achieve a fast and reliable determination of plasma myoglobin concentrations. Precision complied with the recommended analytical performance criteria. Method comparison and recovery experiments indicated, that despite good between-method correlations, the Stratus® CS method overestimated myoglobin concentrations in comparison with values obtained on Cobas® Integra 400 and BN™A. However, since the manufacturers’ cut-off for elevated plasma myoglobin levels was higher for Stratus® CS than for other techniques, few discrepant results were observed between methods. Elevated levels of hemoglobin, triglycerides and rheumatoid factors did not interfere in the Stratus® CS method but hyperbilirubinemia caused a positive difference.Circulerend myoglobine wordt beschouwd als een vroege en gevoelige merker voor acute coronaire aandoeningen. De lange analysetijd beknot echter de klinische bruikbaarheid van myoglobine-assays. We evalueerden de analytische performantie van de op dendrimeertechnologie gebaseerde Stratus® CS fluorometrische enzym-immunoassay, en bevonden de assay als een snelle en betrouwbare bepalingsmethode voor myoglobineconcentraties in plasma. De precisie voldeed aan de aanbevolen analytische performantiecriteria. Methodenvergelijking en recovery-experimenten toonden, ondanks de bevredigende correlaties tussen de verschillende methodes, een overschatting aan van de myoglobineconcentraties bepaald met Stratus® CS, in vergelijking met deze bepaald met Cobas® Integra 400 en BN™A. Daar de door de fabrikant vermelde cut-off voor verhoogde myoglobineconcentraties in plasma hoger was voor Stratus® CS dan voor de andere methoden, werden slechts weinig discrepante resultaten tussen de verschillende methoden teruggevonden. Verhoogde concentraties aan hemoglobine, triglyceriden en reumatoïde factoren, interfereerden niet met de Stratus® CS-methode, doch hyperbilirubinemie veroorzaakte een positief verschil.

Quantitative Analysis of 17β-Estradiol in River Water by Fluorometric Enzyme Immunoassay Using Biotinylated Estradiol

A sensitive and simple immunoassay to determine 17beta-estradiol (E2) in fresh water was developed. The method is based on a solid-phase avidin-biotin binding assay and solid phase extraction. The binding event of E2 to the antibody is detected indirectly by the competitive reaction between E2 and biotinylated estradiol (BE) as a tracer for the limited binding sites of antibodies immobilized onto the wall of a microtiter plate. Namely, E2 concentrations are determined from the strong interaction between BE and avidin conjugated with horseradish peroxidase (avidin-HRP). In order to achieve a sensitive measurement for the binding of BE to the antibody immobilized on the microtiter plate substrate, QuantaBlu fluorogenic peroxidase substrate (QFPS) was employed. The detection limit and the linear range of E2 determination were 27 pM and 27 - 7480 pM, respectively. The relative standard deviations (RSD) for the E2 assay were between 0.3 and 12.0% (n = 3). The cross-reactivities of several other estrogens in this assay system were also investigated. No serious influences from any cross-reaction caused by other estrogens tested in this experiment were observed. The determination of E2 in water samples from eight rivers and a marsh in Hokkaido was performed by the immunoassay combined with solid phase extraction. It was found that the concentration of E2 was in the range between 0.06 and 67 pM.

Zinc, Iron, Copper, Selenium, Lactoferrin, and Ferritin in Human Pus

Restriction of zinc and iron available for microbial growth in tissues are well-recognized host defense mechanisms. The present studies were performed to characterize some constituents of human pus that may affect these important host defenses. Zinc, iron, copper, calcium, and magnesium in pus were measured using an atomic absorption spectrophotometer; selenium was measured fluorometrically. Ferritin was measured with a fluorometric enzyme immunoassay, and lactoferrin was measured with a radial diffusion assay. The growth of Escherichia coli at 37 degrees C was measured in pus supernate adjusted to pH 5.5 or 7.4, in boiled supernate, or in supernate adjusted with 1.3 mM iron or 0.9 mM zinc singly or together. Zinc and iron concentrations in pus exceeded normal serum. Calcium and magnesium levels were 2- to 3-fold lower and higher, respectively, than normal serum values. Lactoferrin concentrations of were 880 +/- 48 microg/mL and ferritin levels were 20,726 +/- 2,667 ng/mL. Growth of an E coli strain was inhibited in pus at pH 5.5 but not at pH 7.4, and growth was enhanced by addition of iron or zinc to E coli suspended in pus at pH 6.7. To our knowledge, this is the first report of the zinc, iron, copper, selenium, lactoferrin, and ferritin levels of human pus. These studies provide additional insight into host defense mechanisms mediated by the restriction of the bioavailability of zinc and iron in suppurative infection.

Ecdysteroid concentrations through various life-stages of the meiobenthic harpacticoid copepod, Amphiascus tenuiremis and the benthic estuarine amphipod, Leptocheirus plumulosus

Endocrine function in arthropods has principally been characterized in insects and malacostracan crustaceans. However, meiofauna represent the most abundant metazoan marine taxa, with harpacticoid copepods comprising the second most abundant taxon. In addition, their diminutive biomass has made characterization of endocrine components difficult, so little is known about endocrine control of reproduction, molting, and growth in meiofauna. In this study, a sensitive fluorometric enzyme immunoassay (EIA) was utilized to quantify and compare the arthropod molting hormone, 20-hydroxyecdysone (20E), in various life-cycle and developmental stages of a laboratory reared meiobenthic copepod, Amphiascus tenuiremis, and in an amphipod, Leptocheirus plumulosus. In copepods, gravid females carrying late stage pre-hatch embryos contained significantly more 20E (390±252 fmol/female) than gravids carrying early (Stage-I) embryos (172±83 fmol/female). In contrast, ecdysteroid levels in Stage-I L. plumulosus gravid females (277±83 fmol/female) was greater than pre-hatch gravid females (146±42). Stage-I embryos of both copepods (19±10) and amphipods (11±5 fmol/embryo) possessed lower ecdysteroid content than copepod (35±15) and amphipod (43±33 fmol/embryo) pre-hatch embryos. Ecdysteroid levels were also assessed in naupliar, juvenile, adult male and non-gravid female copepod life-stages. In addition, ecdysteroids measured in field collected copepod species indicated gravid females possessed ecdysteroid levels similar to gravid A. tenuiremis. However, upon normalization of egg sac 20E content by brood size, embryos from larger broods contained lower levels of ecdysteroids when compared to embryos from smaller clutch sizes-indicating an inverse embryo/ecdysteroid relationship may exist across species.

NON-RADIOMETRIC IMMUNOASSAYS [FLUOROIMMUNOASSAY (FIA) AND FLUOROMETRIC ENZYME IMMUNOASSAY (FEIA)] WITH RADIOIMMUNOASSAY (RIA) FOR EVALUATION OF ADRENAL FUNCTION IN NORMAL AND HYPERCORTISOLEMIC DOGS

Non-radiometric immunoassays offer many advantages over radiometric assays, such as higher stability of kit compounds and absence of potential hazardous effects for users and environment. The comparison of cortisol measurements by fluoroimmunoassay (FIA) and fluorometric enzyme immunoassay (FEIA) with radioimmunoassay (RIA) in adrenal function evaluation of normal (n=50) and hypercortisolemic dogs (n=12) was proposed. Serum concentrations of cortisol were measured in basal conditions and 8 hours after dexamethasone (DEX) suppression (0.01mg/kg/IV). All our reference values were based on the 5th and 95th percentile. The values for basal cortisol of healthy dogs were 0.20 to 2.35mug/d&lt;img SRC="http:/img/fbpe/cr/v32n2/a12img01.gif"&gt; for FIA, 0.30 to 5.39mug/d&lt;img SRC="http:/img/fbpe/cr/v32n2/a12img01.gif"&gt; for FEIA, and 0.65 to 4.64mug/d&lt;img SRC="http:/img/fbpe/cr/v32n2/a12img01.gif"&gt; for RIA. After DEX suppression the values were &lt;0.87mug/d&lt;img SRC="http:/img/fbpe/cr/v32n2/a12img01.gif"&gt;, &lt;0.30mug/d&lt;img SRC="http:/img/fbpe/cr/v32n2/a12img01.gif"&gt; and &lt; 0.80mug/d&lt;img SRC="http:/img/fbpe/cr/v32n2/a12img01.gif"&gt; for FIA, FEIA and RIA, respectively. In hypercortisolemic dogs, the values of cortisol (mean ± SD) in basal and post-DEX conditions were 2.71 + 0.41mug/d&lt;img SRC="http:/img/fbpe/cr/v32n2/a12img01.gif"&gt; and 1.73 + 1.15mug/d&lt;img SRC="http:/img/fbpe/cr/v32n2/a12img01.gif"&gt; for FIA, 7.05 + 2.85mug/d&lt;img SRC="http:/img/fbpe/cr/v32n2/a12img01.gif"&gt; and 4.93 + 2.26mug/d&lt;img SRC="http:/img/fbpe/cr/v32n2/a12img01.gif"&gt; for FEIA, and 4.80 + 1.43mug/d&lt;img SRC="http:/img/fbpe/cr/v32n2/a12img01.gif"&gt; and 3.52 + 1.08mug/d&lt;img SRC="http:/img/fbpe/cr/v32n2/a12img01.gif"&gt; for RIA. Statistically significant differences (p&lt;0.05) between the normal and the hypercortisolemic groups (Kruskal-Wallis test) were observed in the three methods, and between basal and post-DEX values (Wilcoxon test) using RIA and FEIA methods but not with FIA. Cortisol determinations by FEIA and RIA methods at DEX suppression test showed 100% of sensitivity and specificity for the diagnosis of hyperadrenocorticism in dogs. The results demonstrate that serum cortisol concentrations measurements by FEIA is a suitable alternative to the traditional RIA method for adrenal function evaluation in dogs.

Predictive value of perioperative cardiac troponin I for adverse outcome in coronary artery bypass surgery.

Cardiac Troponin I (cTnI) is a well-known marker for myocardial damage in patients undergoing aorto-coronary bypass grafting (CABG) peaking 6-8 h after aortic declamping. The aim of this study was to evaluate cTnI release in the course of CABG procedures early, i.e. after the cessation of cardiopulmonary bypass (CPB) in order to recognize unstable cardiac function leading to hemodynamic deterioration and resulting in an adverse outcome (AO). AO is defined as the onset of myocardial infarction and/or death peri/postoperatively. Five-hundred and forty consecutive patients who underwent CABG were evaluated for cTnI release immediately prior to the induction of anesthesia (IND) and after termination of CPB (END). Standard CPB with ante/retrograde cold blood cardioplegia was used. Patients with any of the following criteria were excluded: (1), CABG within 7 days of myocardial infarction; (2), emergency operation for both unstable angina and for coronary occlusion at angioplasty; (3), CABG with concomitant surgical cardiac procedures; (4), preoperative renal dysfunction requiring hemodialysis; (5), redos. Troponin I was measured with the Stratus CS fluorometric enzyme immunoassay analyzer (Dade-Behring) running on site in the operation room (OR), so values of cTnI could be obtained within 15 min. There were six deaths (1.1%) in the entire series, Q-wave myocardial infarction occurred in 19 patients (3.5%), AO was experienced by 21 patients (3.9%). The mean preoperative cTnI level was 0.04+/-0.17 ng/l (mean+/-standard deviation) for the entire group. The END cTnI level for the AO-group was 0.91+/-0.5 ng/l; for all other patients, this was 0.37+/-0.3 ng/l (P<0.001). Changes in intraoperative cTnI levels relative to time course showed a marked increase for the AO-group (0.0038+/-0.0035 ng/l*min) as compared with non-AO patients (0.0019+/-0.0015 ng/l*min; P=0.028). The receiver operating characteristic curve indicates a cTnI level at CPB-end of higher than 0.495 ng/l with an area under the curve of 0.83 as the optimal cut-off point for predicting AO with a sensitivity and specificity of 76.2%. Stepwise logistic regression analysis revealed END cTnI level (odds ratio, 17.24; P<0.001), CPB time (odds ratio, 1.03; P=0.001), female sex (odds ratio, 3.8; P=0.011) as significant independent predictors for AO. Age of over 70 years (P=0.8), Cleveland Clinic risk score (P=0.65), diabetes (P=0.26), elevated preoperative creatinine level (P=0.77), severe left ventricular dysfunction (P=0.51), the number of grafts performed (P=0.15), and change of intraoperative cTnI level relative to time course (P=0.94) did not reach statistical significance. cTnI release as determined at the end of CABG procedures represents a strong predictor of an AO after surgery. Analyzing blood samples for cTnI with an automated device on site in the OR provides for immediate results, so specific diagnostic and therapeutic interventions can be performed before hemodynamics deteriorate.

Chronic fatigue syndrome: circadian rhythm and hypothalamic-pituitary-adrenal (HPA) axis impairment.

Chronic Fatigue Syndrome (CFS) is a clinical condition characterized by a persistent or relapsing debilitating fatigue at rest, lasting more than 6 months, and made worse by exercise. At the present moment, there are three potential etiopathogenic factors: immunologic, viral and neuroendocrine. The purpose of our study was to evaluate possible alterations of the hypothalamic-pituitary-adrenal (HPA) axis in our CFS patients by studying the circadian rhythms of prolactin (PRL), thyrotropic hormone (TSH), adrenocorticotropic hormone (ACTH), and cortisol (CS). A total of 36 patients were enrolled according to the Centers for Disease Control and Prevention case-definition criteria. Twenty healthy subjects were included as controls. Blood samples were taken every 4 hours during a single 24-hour period. We performed a fluorometric enzyme immunoassay with serum PRL, cortisol and TSH, and an immunoradiometric assay with plasma ACTH. The circadian rhythms of PRL, TSH, ACTH and CS were statistically significant in both CFS and control groups. At 24:00 and 04:00 hrs the CFS patients showed lower ACTH levels than healthy subjects (p &#x003C; 0.001); the PRL levels were higher at 04.00 h in CFS patients than in healthy subjects.

Effect of elevated concentration of alkaline phosphatase on cardiac troponin I assays.

Troponin I is the regulatory subunit of troponin complex associated with the actin thin filament within muscle cells. Cardiac troponin I (cTnI) is a good marker for diagnosis of myocardial damage. Several immunoassays are available for determination of cTnI in serum. The Stratus cTnI fluorometric enzyme immunoassay (Dade International) uses alkaline phosphatase (ALP) substrate. The microparticle enzyme immunoassay (MEIA) for cTnI (Abbott Laboratories) also uses ALP conjugate. On the other hand, the chemiluminescent assay (CLIA) for cTnI (Bayer Diagnostics) does not use ALP. ALP activity may frequently be elevated in serum of patients being evaluated for suspected myocardial infarction. Therefore, we studied the potential interference of ALP in cTnI assays. Serum pools were prepared from patients, and various concentrations of ALP solution were added to different aliquots. The cTnI concentrations were measured by the Stratus, MEIA, and CLIA assays. We observed no interference of ALP in the MEIA and CLIA assay for cTnI. On the other hand, we observed significant positive interference of ALP when cTnI concentrations were measured using the Stratus.

Comparison of Cardiac Troponin I Measurements on Whole Blood and Plasma on the Stratus CS Analyzer and Comparison with AxSYM

Assays for cardiac troponin T and troponin I (cTnI) are powerful tools for early identification of patients with acute myocardial infarction (AMI) and for pointing out subjects at highest risk among patients with unstable angina pectoris (1)(2)(3). These applications require increasingly rapid and more sensitive tests. To this end, one of the most commonly used automated assays for cTnI (Stratus® II; Dade) has undergone extensive revision (4)(5). A second-generation quantitative radial partition immunoassay is now used in combination with the Stratus CS fluorometric analyzer, a system designed to meet the needs of STAT and point-of-care testing. The Stratus CS yields cTnI results that are clinically consistent with those obtained with the Stratus II (6). The Stratus CS uses closed routine sample tubes containing anticoagulated whole blood (lithium heparin). The system takes ≤14 min to centrifuge the tube and perform the analysis. The Stratus CS can also manage precentrifuged plasma samples. In the present study, the Stratus CS was used to determine cTnI on whole blood and on preprocessed plasma samples drawn from the same patients. Our purpose was to check the similarity of the results obtained for both samples in the entire measuring range. We also aimed at comparing results from the Stratus CS with cTnI results yielded by the Abbott AxSYM analyzer, with special attention to concentrations below the threshold for AMI. The Stratus CS is a fluorometric enzyme immunoassay analyzer for quantitative determination of the cardiac markers creatine kinase-MB mass, myoglobin, and cTnI (Dade Behring). The test system uses radial partition immunoassay technology, which has been enhanced through the use of a monoclonal capture antibody coupled to Starburst® dendrimers (7). According to the insert for the cTnI method, coronary risk increases at cTnI concentrations >0.4 μg/L, and this value …

High prevalence of congenital toxoplasmosis in Brazil estimated in a 3-year prospective neonatal screening study.

A pilot neonatal screening programme revealed a high (approximately 1 per 4800 live births) prevalence of congenital toxoplasmosis (CT) in the State of Rio Grande do Sul, Brazil. The purpose of this paper was to estimate in a larger prospective study the prevalence of CT in the country. At the beginning of the study, an in-house indirect enzyme immunoassay (EIA) was used, to be later replaced with a commercial capture IgM fluorometric enzyme immunoassay (FEIA). Both methods detect specific anti-Toxoplasma gondii IgM-class antibodies eluted from dried blood spots. Of the total of 140,914 samples received from all over the country, 47 cases were identified and confirmed as CT. This finding suggests a prevalence of 1 per 3000 live births. Of the 47 patients, only eight (17%) had clinical manifestations: two had intracranial calcifications, four had retinal scars, one had an intracranial calcification and retinal scars, and one had hepatosplenomegaly with lymphoadenopathy. The testing was paid for by the patients' families who volunteered for the study and gave their informed consent. The 3-year prospective study using sensitive detection methods, reliable confirmation, and feedback from clinicians showed that CT has an extraordinarily high prevalence in Brazil, in fact the highest ever reported in the world. Although the long-term efficacy of treatment of CT has not been well documented, in view of the availability of reliable diagnostics, confirmation and monitoring, functional logistics, and networking for screening, the insidious nature of the sequelae and the very high prevalence of the disease, neonatal screening for CT should be considered an alternative to no screening at all.

Rapid Measurement of Cardiac Markers on Stratus CS

Biochemical markers of myocardial damage are of fundamental importance in ruling in and ruling out diagnostic strategies for acute coronary diseases, particularly when electrocardiographic findings do not allow a diagnosis. An increasing body of evidence has demonstrated the value of strategies based on specific (cardiac troponin I or T) and sensitive [myoglobin (Myo)] assays in the diagnosis (1), prognosis, and monitoring (2) of patients with acute coronary syndrome. The impact of laboratory information on the management of patients has led to the development and definition of recommendations designed to reduce the therapeutic turnaround time, thus improving the medical outcome for patients. Because of the delay often associated with transport-related problems, the concept of point-of-care testing has been introduced for the measurement of cardiac markers (3). Some requirements, which seem to be of value in assuring quality for devices and diagnostic systems designed for point-of-care testing of cardiac markers, require whole blood or heparinized specimens for effective therapeutic turnaround time and must guarantee quantitative, accurate, and precise results comparable to those provided by central laboratories. The aim of our study, therefore, was to evaluate the analytical performance of the Stratus CS (SCS; Dade Behring), a fluorometric enzyme immunoassay analyzer based on solid-phase radial partition immunoassay technology, for quantitative measurement of creatine kinase isoenzyme MB mass concentration (CK-MB), Myo, and troponin I (TnI) in whole blood samples collected using lithium heparinate as anticoagulant. The samples may be processed automatically by the analyzer, which provides for centrifugation on board before the analytical phase, or heparinized plasma may be dispensed directly into a sample cup for analysis. Briefly, after the first antibody is added to a glass fiber paper linked to the dendrimer, the sample and the second antibody are pipetted. Finally, enzyme activity is started by the substrate wash solution with the simultaneous …

IS CARDIAC TROPONIN I A RELIABLE MARKER TO ASSESS MYOCARDIAL INJURY AFTER PEDIATRIC CARDIAC SURGERY?

Introduction: Cardiac troponin I (CTnI) as been used as a specific marker to assess the postoperative myocardial injury in congenital heart diseases [1]. The aim of our study was to evaluate in a large cohort of pediatric patients how CTnI (in association with other techniques) could assess the postoperative myocardial injury and the severity of the early postoperative outcome. Methods: A group of 263 consecutive patients (mean 8.2 years), undergoing cardiac surgery with cardiopulmonary bypass and aortic cross clamping for congenital heart diseases was studied. All patients received the same protocol of anesthesia, management of CPB and myocardial protection. CTnI was measured routinely by the Stratus fluorometric enzyme immunoassay technique (Dade). Blood samples were collected after induction of anesthesia, 15 minutes and 3 hours after aortic unclamping and then every 6 hours during 45 hours. After the procedure and during 45 hours, the patients were enrolled in three groups after meeting criteria of severity of myocardial dysfunction. Group 1: unchanged EKG, unchanged ventricular kinesia on TTE and dobutamine or dopamine requirement < 5 mcg/kg/min. Group 2: modified EKG with Q wave or ST segment changes or arrhythmias or conduction abnormalities, echocardiography with ventricular hypokinesia and dobutamine or dopamine requirement > 5 mcg/kg/min. Group 3: modified EKG (same than Gr. 2), echocardiography with ventricular akinesia, and dobutamine or dopamine requirement > 5 mcg/kg/min associated with epinephrine associated or not with delayed sternal closure. For the comparison of the mean value between two groups, two-tailed Student t-test and Z-test were used. A probability of less than 0.05 was considered significant. Results: 185 patients (70%) were assigned group 1, 38 (14.4%) patients group 2 and 40 (15.2%) patients group 3. Mean CTnI plasma levels of groups 1, 2 and 3 were not statistically different in the preoperative period (first sample) and 15 minutes after aortic unclamping. From H 3 to H 45, mean CTnI plasma levels of group 1 were statistically different from groups 2 and 3 (p<0.01). Mean CTnI plasma levels were not statistically different between groups 2 and 3. The profiles of the perioperative CTnI plasma level time course are similar in the three groups and peaked between H 15 and H 21. In group 3, the peak of CTnI plasma level is 2.1 times as elevated as in group 1 at H 3 and 2.48 times at H 21. There was no mortality in group 1 and 2, and 26 deaths (9.8%) occured in group 3. Discussion: Any procedure for correction of congenital heart disease may result in postoperative myocardial dysfunction related to the surgical technique, the cardiopulmonary bypass and/or the myocardial protection. In this study, the CTnI plasma level time course during the first 45 hours compared with the severity score is related with the severity of the myocardial dysfunction. The predictive value of a high concentration of CTnI during the nine first hours suggested by Taggart [1], is demonstrated with the occurence of the whole mortality in the group 3 where the CTnI plasma levels are the more elevated during the first 45 hours. Conclusion: An early post-operative increase of CTnI plasma level, associated with an echocardiographic myocardial dysfunction allow to apply the adequate therapy before any clinical sign of hemodynamic failure appear. Figure 1Figure 1