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New neonatal thyrotropin enzyme immunoassay with fluorimetric detection: Comparison with time-resolved fluoroimmunoassay
Abstract
This short communication compares a novel fluorimetric microplate enzyme immunoassay (FEIA) with a commercial time-resolved fluoroimmunoassay for the determination of thyrotropin in dried blood spots. The evaluation was performed using a retrospective study design with newborn blood samples from three screening centres. Non-parametric Spearman rank correlation analysis revealed highly significant positive correlation between methods: rs = 0.465, p < 0.0001 (Hannover), rs = 0.659, p < 0.0001 (Minsk), rs = 0.755, p < 0.0001 (Helsinki). Wilcoxon signed rank test performed for paired FEIA and time-resolved fluoroimmunoassay showed that the results obtained by both tests represented the same distribution (p < 0.0001). The new method, using fluorimetric detection, can be performed with the instrumentation commonly used for the screening of congenital hypothyroidism and phenylketonuria. Results are obtained within three to four hours after arrival of the sample in the laboratory. Preliminary evaluation indicates the method to be a suitable alternative to time-resolved fluoroimmunoassay for neonatal thyroid function screening.
... In Deutschland wurde die Hypothyreose 1978 als Zielkrankheit in das Neugeborenen-Screening aufgenommen. Als technische Varianten wurden Immunoassays entwickelt, die ohne Verwendung radioaktiver Indikatoren mit farbbildenden Enzymreaktionen oder Fluoreszenzsignalen arbeiten [10]. ...
Zusammenfassung
Seit mehr als 5 Jahrzehnten wird allen Neugeborenen in Deutschland eine Vorsorgeuntersuchung zur Früherkennung angeborener behandelbarer Krankheiten angeboten. Seit Beginn sind so etwa 35 Mio. Kinder untersucht worden.
Anfangs ging es nur um die Früherkennung der Phenylketonurie, die ohne frühzeitige Behandlung zu nicht mehr korrigierbarer geistiger Behinderung führt. Der bakteriologische Guthrie-Test erlaubte den Nachweis erhöhter Konzentrationen von Phenylalanin. Die heute eingesetzten Methoden sind das Ergebnis einer über Jahrzehnte verlaufenden Entwicklung. Hinzugekommen sind Tests zur Bestimmung von Enzymaktivitäten, Immunoassays zur Früherkennung wichtiger hormoneller Störungen wie der angeborenen Schilddrüsenunterfunktion sowie Hochdruck-Flüssigkeits-Chromatografie zur Identifizierung pathologischer Hämoglobine. Die sehr anspruchsvolle Tandem-Massenspektrometrie ermöglicht die gleichzeitige Erfassung von Aminosäuren und Derivaten organischer Säuren und Fettsäuren. Auch Steroide können damit identifiziert werden. Die Spezifität lässt sich durch Kombination mit chromatografischer Vortrennung noch erhöhen. In den letzten Jahren wurden die chemisch-analytischen Untersuchungen ergänzt durch gendiagnostische Verfahren, wie beispielsweise quantitative oder qualitative Polymerasekettenreaktion (PCR).
Der Stand der Labortechnik ist keineswegs endgültig. Sowohl die klassische Analytik als auch besonders die genetischen Verfahren stehen vor einer weiteren rasanten Entwicklung. Während die Ausweitung des Screenings auch eine Folge der technischen Entwicklung ist, hängt die Einbeziehung weiterer angeborener Erkrankungen grundsätzlich von einer jeweiligen Therapie ab. Aber gerade hier werden gegenwärtig viele Neuerungen erprobt. Im Vordergrund des Interesses steht dabei die Gentherapie.
... The aim of NS programs is to detect all cases with the disease as early as possible, with an acceptable cost-benefit ratio and to avoid false positive results. In recent years, more sensitive and automated methods (chemiluminescence, fluoroimmunoassay, etc.) for determining both TSH and T4 in dried blood spots have been introduced (16,17,18,19,20,21). These new methods have increased sensitivity and specificity in the detection of CH. ...
Newborn screening (NS) for congenital hypothyroidism (CH) is one of the major achievements in preventive medicine. Most neonates born with CH have normal appearance and no detectable physical signs. Hypothyroidism in the newborn period is almost always overlooked, and delayed diagnosis leads to the most severe outcome of CH, mental retardation, emphasizing the importance of NS. Blood spot thyroid stimulating hormone (TSH) or thyroxine (T4) or both can be used for CH screening. The latter is more sensitive but not cost-effective, so screening by TSH or T4 is used in different programs around the world. TSH screening was shown to be more specific in the diagnosis of CH. T4 screening is more sensitive in detecting especially those newborns with rare hypothalamic-pituitary-hypothyroidism, but it is less specific with a high frequency of false positives mainly in low birth weight and premature infants. The time at which the sample is taken may vary. In the majority of the centers, blood is obtained from a heel prick after 24 hours of age to minimize the false positive high TSH due to the physiological neonatal TSH surge that elevates TSH levels and causes dynamic T4 and T3 changes in the first 1 or 2 days after birth. Early discharge of mothers postpartum has increased the ratio of false positive TSH elevations. Although transient hypothyroidism may occur frequently, all these infants should be treated as having CH for the first 3 years of life, taking into account the risk of mental retardation. A reevaluation after 3 years is needed in such patients. The goal of initial therapy in CH is to minimize neonatal central nervous system exposure to hypothyroidism by normalizing thyroid function, as rapidly as possible.
Conflict of interest:None declared.
Modern medical practices have made great progress in the prevention and management of diseases through early diagnosis and treatment methods. At the center of these developments are newborn and childhood screening programs. These screening programs have been developed in order to contribute to children living a healthy life starting from birth, to identify them at an early stage before signs and symptoms of disease appear, and to make the necessary interventions in a timely manner. This book in your hands aims to comprehensively cover the most up-to-date information and practices on newborn and childhood screening programs. This work, which has been meticulously prepared by authors who are experts in the eld, covers a wide range of topics such as clinical practices, scientic foundations of screening tests, organization of programs and ethical dimensions. We believe this book will be a valuable resource for pediatricians, family physicians, public health professionals, nurses, and medical students on their professional journeys. It will help health professionals improve their knowledge and skills and contribute to our children having a healthy future. We hope that your interest in newborn and childhood screening programs and your devoted work on this subject will lead to the raising of healthier generations. With love and respect.
The technical and diagnostic performance of fully automated immunoassays for free thyroxine and thyrotropin using streptavidin/biotin technology (Enzymun-Test) were examined. The between-assay precision for free thyroxine was 10.4%, 5.4%, 2.5%, 2.3%, 1.1%, and 1.8% at 3.02, 6.27, 17.2, 21.9, 25.6, 42.7 pmol/l; and for thyrotropin was 14.2%, 4.7%, 2.9%, 2.8%, 3.2%, 4.5% at 0.12, 0.46, 1.03, 2.05, 4.8, 12.7 mU/l. The functional detection limit of the assay was 0.09 mU/l. Results for the free thyroxine method correlated well with the IMx (r = 0.91) and the equilibrium dialysis (r = 0.95) assay. Results for the thyrotropin method correlated well with the Tandem-TSH (r = 0.99) and the IMx (r = 0.99) assays. The euthyroid reference range was 11-23 pmol/l and 0.5-3.9 mU/l for free thyroxine and thyrotropin respectively. The free thyroxine assay was not influenced by changes in albumin or thyroxine binding globulin concentration but showed increases at oleic acid concentrations > 4 mmol/l. Spuriously elevated free thyroxine concentration were found in 4 patients, due to assay interference by antibodies in the serum. In a follow up study of 46 patients with non-thyroidal illness, serial measurements showed fluctuating free thyroxine and thyrotropin concentrations with abnormal results occurring in 34%. In a hospital setting, a wider range of free thyroxine (10-28 pmol/l) and thyrotropin (0.22-5.9 mU/l) concentration may be observed in patients who are clinically euthyroid. Abnormal thyroid function tests were however transient and follow up resolved most diagnostic problems.
An immunoradiometric assay (IRMA) of human thyrotropin (hTSH), based on magnetic solid phase separation, was studied especially in terms of its nonspecific bindings (B0) which were identified as a product of the interaction between an altered form of radioiodinated anti-hTSH monoclonal antibody (125I-mAB) and the uncoupled magnetizable cellulose particle (matrix). Preincubation with the same matrix, solid phase saturation with milk proteins, tracer storage at 4 degrees C and serum addition during incubation were found to be particularly effective in preventing their formation. These findings were used to reproducibly decrease nonspecific bindings to values < 0.1% (or < 70 cpm), thus increasing the signal-to-noise ratio (B60/B0) up to values of 300-500. This way hTSH radioassays were obtained with functional sensitivities of about 0.05 mIU/L and analytical sensitivities of the order of 0.02 mIU/L. Such sensitivities, and, more importantly, a general improvement in assay performance, were obtained in a highly reproducible manner and all over the useful tracer life.
Objectives: to establish a database of literature and other evidence on neonatal screening programmes and technologies for inborn errors of metabolism. To undertake a systematic review of the data as a basis for evaluation of newborn screening for inborn errors of metabolism. To prepare an objective summary of the evidence on the appropriateness and need for various existing and possible neonatal screening programmes for inborn errors of metabolism in relation to the natural history of these diseases. To identify gaps in existing knowledge and make recommendations for required primary research. To make recommendations for the future development and organisation of neonatal screening for inborn errors of metabolism in the UK.
How the research was conducted: there were three parts to the research. A systematic review of the literature on inborn errors of metabolism, neonatal screening programmes, new technologies for screening and economic factors. Inclusion and exclusion criteria were applied, and a working database of relevant papers was established. All selected papers were read by two or three experts and were critically appraised using a standard format. Seven criteria for a screening programme, based on the principles formulated by Wilson and Jungner (WHO, 1968), were used to summarise the evidence. These were as follows. Clinically and biochemically well-defined disorder. Known incidence in populations relevant to the UK. Disorder associated with significant morbidity or mortality. Effective treatment available. Period before onset during which intervention improves outcome. Ethical, safe, simple and robust screening test. Cost-effectiveness of screening. A questionnaire which was sent to all newborn screening laboratories in the UK. Site visits to assess new methodologies for newborn screening. The classical definition of an inborn error of metabolism was used (i.e., a monogenic disease resulting in deficient activity in a single enzyme in a pathway of intermediary metabolism).
Research findings: inborn errors of metabolism. Phenylketonuria (PKU) (incidence 1:12,000) fulfilled all the screening criteria and could be used as the 'gold standard' against which to review other disorders despite significant variation in methodologies, sample collection and timing of screening and inadequacies in the infrastructure for notification and continued care of identified patients. Of the many disorders of organic acid and fatty acid metabolism, a case can only be made for the introduction of newborn screening for glutaric aciduria type 1 (GA1; estimated incidence 1:40,000) and medium-chain acyl CoA dehydrogenase (MCAD) deficiency (estimated incidence 1:8000-1:15,000). Therapeutic advances for GA1 offer prevention of neurological damage but further investigation is required into the costs and benefits of screening for this disorder. MCAD deficiency is simply and cheaply treatable, preventing possible early death and neurological handicap. Neonatal screening for these diseases is dependent upon the introduction of tandem mass spectrometry (tandem MS). This screening could however also simultaneously detect some other commonly-encountered disorders of organic acid metabolism with a collective incidence of 1:15,000
The ability to isolate and measure multiple complex analytes in a single biological sample holds great potential in many biomedical fields, especially immunology and diagnostic clinical chemistry. We have developed a procedure involving recycling immunoaffinity chromatography for the simultaneous measurement of a number of analytes in a single sample. The procedure is based on the passage of a fluorochrome-labelled sample through a battery of small immunoaffinity columns, each column extracting a single analyte. Detection is achieved by acid elution of the bound analytes and laser-induced fluorescence. We have applied this system to a number of different biological fluids and found that it is capable of reliably isolating and measuring up to ten different cytokines in a 25-microl sample of human body fluid.
Newborn screening for congenital hypothyroidism (CH) is one of the major achievements of preventive medicine, as the condition occurs frequently (1/4000 newborns) and results in brain damage if not detected and treated in the first few days of life. Measurement of T4 and/or TSH in dried blood spots collected on the second through fifth days of life are the most widely used methods in screening programs for CH currently. Some children with the disease may be missd in any screening program, however, owing to factors related to the disease itself and the methods employed in its detection, as well as factors ascribed to the element of human error, ie screening errors. The methods employed in newborn screening programs for CH, their efficiency in disease detecetion, and biological factors as well as screening errors leading to missed cases are discussed.
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