Generation and characterization of monoclonal antibodies against thyroid-stimulating hormone for newborn screening of congenital hypothyroidism.

Congenital hypothyroidism (CH) is one of the most frequent inherited-metabolic diseases in the world, and the main cause of treatable mental retardation in children. Because signs and symptoms of this disease are often scarce and not easily recognizable, newborns are screened for the early CH detection at birth. The Center of Immunoassay (CIE) has developed the UMELISA® TSH Neonatal and UMELISA® TSH to determine neonatal thyroid-stimulating hormone (TSH) levels in dried blood and serum samples. Both reagent kits use the same polystyrene plates coated with anti-ß-TSH monoclonal antibodies (MAbs), but one of these is commercially acquired. Obtaining appropriate anti-TSH MAbs at the CIE would guarantee economic independence and security in the production of these kits. Immunization of mice with TSH led to the generation of 7G11E3, an anti-ß-TSH IgG1-secreting hybridoma. The high affinity of 7G11E3 MAb and its characteristic epitopic recognition explain its better performance when adsorbed to UMELISA® plates for capturing low amounts of TSH in comparison with the studied MAbs. Performance of assays using polystyrene plates coated with 7G11E3 MAb was studied. Recovery percentages (100.0-106.7% for UMELISA® TSH NEONATAL and 97.3-99.0% for UMELISA® TSH) and intra (5.2-7.9% for UMELISA® TSH NEONATAL and 3.2-5.3% for UMELISA® TSH) and inter (6.6-7.7% for UMELISA® TSH NEONATAL and 5.2-8.0% for UMELISA® TSH) coefficients of variation were similar to the ones described for the commercial kits. Limits of detection and quantification were 1.0 and 3.8 mIU/L for UMELISA® TSH NEONATAL, and 0.3 and 0.6 mIU/L for UMELISA® TSH, respectively. The results also showed high overall concordance between assays (n = 2 019, ρc = 0.90 for UMELISA® TSH NEONATAL and n = 200, ρc = 0.94 for UMELISA® TSH). The 7G11E3 MAb meets the requirements for its use in the plates of UMELISA® TSH kits for CH newborn screening in Cuba. Abbreviations: CECMED, Center for the State Control of Medicaments and Medical Equipment and Devices; CH, congenital hypothyroidism; CIE, Center of Immunoassay; CLSI, Clinical and Laboratory Standards Institute; CV coefficient of variation; DBS, dried blood spots; LOB, limit of blank; LOD, limit of detection; LOQ, limit of quantitation; SD, standard deviation; Sr, repeatability standard deviation; SUMA, Ultra Micro Analytic System; UMELISA, ultramicro enzyme-linked immunosorbent assay.

Foetal thyroid dysfunction: treat the mother first!

A case is presented of foetal compensated hypothyroidism due to persisting low maternal serum FT4 at the beginning of pregnancy. Diagnosis was made by means of foetal ultrasound followed by foetal blood sampling because of atypical findings. Foetal thyroid hypertrophy resolved progressively as exogenous thyroxine was administered to the mother. This case highlights once again the importance of adequate thyroid function during pregnancy.

Severe unsuspected maternal hypothyroidism discovered after the diagnosis of thyrotropin receptor-blocking antibody-induced congenital hypothyroidism in the neonate: failure to recognize and implications to the fetus.

BACKGROUND: Whereas most adequately treated children with congenital hypothyroidism (CH) do well neurodevelopmentally, when both the maternal and fetal thyroid glands are compromised, significant cognitive delay can occur despite early and aggressive postnatal therapy. Maternal thyrotropin-stimulating hormone receptor (TSHR)-blocking antibodies (Abs) can be transmitted to the fetus and cause combined maternal-fetal hypothyroidism. Current guidelines recommend their measurement only if mothers have known autoimmune thyroid disease, there is a history of a previously affected sibling, or when transient CH is suspected. RESULTS: We report 3 infants in whom the diagnosis of maternal hypothyroidism was not known and was identified only after CH was diagnosed in their babies. One of these infants had developmental delay despite rapid normalization of thyroid function postnatally. All 3 mothers had potent TSHR Abs in serum, but thyroid peroxidase Abs and thyroglobulin Abs were detectable in only 2 of them. CONCLUSIONS: TSHR-blocking Ab-induced CH should be suspected in any baby with CH irrespective of the known family history, especially if the hypothyroidism is severe and a eutopic thyroid gland is demonstrated on imaging. Measurement of TSHR Abs is necessary to establish the diagnosis; the presence of other thyroid Abs is insufficiently sensitive and may miss some cases.

The IGSF1 deficiency syndrome: characteristics of male and female patients.

CONTEXT: Ig superfamily member 1 (IGSF1) deficiency was recently discovered as a novel X-linked cause of central hypothyroidism (CeH) and macro-orchidism. However, clinical and biochemical data regarding growth, puberty, and metabolic outcome, as well as features of female carriers, are scarce. OBJECTIVE: Our objective was to investigate clinical and biochemical characteristics associated with IGSF1 deficiency in both sexes. METHODS: All patients (n = 42, 24 males) from 10 families examined in the university clinics of Leiden, Amsterdam, Cambridge, and Milan were included in this case series. Detailed clinical data were collected with an identical protocol, and biochemical measurements were performed in a central laboratory. RESULTS: Male patients (age 0-87 years, 17 index cases and 7 from family studies) showed CeH (100%), hypoprolactinemia (n = 16, 67%), and transient partial GH deficiency (n = 3, 13%). Pubertal testosterone production was delayed, as were the growth spurt and pubic hair development. However, testicular growth started at a normal age and attained macro-orchid size in all evaluable adults. Body mass index, percent fat, and waist circumference tended to be elevated. The metabolic syndrome was present in 4 of 5 patients over 55 years of age. Heterozygous female carriers (age 32-80 years) showed CeH in 6 of 18 cases (33%), hypoprolactinemia in 2 (11%), and GH deficiency in none. As in men, body mass index, percent fat, and waist circumference were relatively high, and the metabolic syndrome was present in 3 cases. CONCLUSION: In male patients, the X-linked IGSF1 deficiency syndrome is characterized by CeH, hypoprolactinemia, delayed puberty, macro-orchidism, and increased body weight. A subset of female carriers also exhibits CeH.

An immunoglobulin G complexed form of thyroid-stimulating hormone (macro thyroid-stimulating hormone) is a cause of elevated serum thyroid-stimulating hormone concentration.

BACKGROUND: Macrocomplexes can be the cause of elevated serum hormone concentrations and may cause diagnostic confusion. This is well recognized for prolactin and commonly screened for using polyethylene glycol (PEG) precipitation. The phenomenon and a suitable screening method is less familiar with respect to thyroid-stimulating hormone (TSH). METHOD: Samples sent to the laboratory for routine analysis of thyroid function and found to have a TSH >10 mU/L were evaluated to determine the prevalence of macro-TSH in the Roche Elecsys assay, using PEG precipitation with confirmation by gel filtration chromatography. RESULTS: Of 495 samples tested, 3 (0.6%) were found to have macro-TSH. From the distribution of recoveries, a cut-off <25% was determined for identifying samples requiring further investigation for the presence of macro-TSH. CONCLUSION: The prevalence of elevated TSH due to macro-TSH was found to be 0.6%. Laboratories should be aware of this cause of assay interference.

The natural history of the hyperthyrotropinemia of children born prematurely.

OBJECTIVE: Non-autoimmune hyperthyrotropinemia has been previously reported among children born prematurely. The aim of this study was to follow up their thyroid function, volume, and structure and to investigate the relationship with growth, IGF-I, lipid profile, and insulin sensitivity. METHODS: Seventy-two children born prematurely (33.2±2.2 weeks), 26 appropriate (AGA) and 46 small for gestational age (SGA), were evaluated at the age of 7.6±2.3 yr (time 1) and at the age of 11.4±2.3 yr (time 2). We also measured TSH, free T(3) (fT(3)), free T(4) (fT(4)), thyroperoxidase antibodies (TPO-Ab), thyroglobulin antibodies (TG-Ab), thyroid ultrasound, auxological parameters, lipid profile, glucose, and insulin level. RESULTS: In the AGA group TSH was similar in both times (2.7±1.0 vs 3.0±0.9 mU/l) and above the upper normal limit in 4 (15.4%) subjects at time 1 and in 6 (23.7%) subjects at time 2 (ns). In the SGA group, TSH was similar in both times (2.8±1.2 vs 2.5±1.0 mU/l) and above the upper normal limit in 11 (23.9%) subjects at time 1 and 5 (10.8%) subjects at time 2 (ns). fT(4) and fT(3) were always normal and TPO- and TG-Ab absent. Thyroid volume increased progressively, but significantly only in the AGA group (p=0.0005). The thyroid structure was always normal and there was no influence on the growth and the biochemical profile. CONCLUSIONS: Some ex-premature babies show a mild and variable thyroid dysfunction, which does not seem to evolve toward an overt thyroid dysfunction.

Transient congenital hypothyroidism due to thyroid-stimulating hormone receptor blocking antibodies: a case series.

We describe seven infants with transient congenital hypothyroidism (CH) due to maternal thyroid-stimulating hormone receptor (TSH-R) blocking antibodies (TRAb) identified over three decades of newborn screening for CH in Wales, UK that represents a minimum incidence of 1.6% of CH cases. Infants with transient CH due to maternal TRAb presented with a spectrum of clinical and biochemical hypothyroidism. Blood spot TSH concentrations ranged 60.5-332 mIU/L. CH was confirmed by plasma thyroid function tests in all cases (plasma TSH ranged 21-752 mIU/L). The seven infants belonged to five different families. On examination, four infants were clinically hypothyroid. Five infants had a thyroid ultrasound, of which three were abnormal. All infants were treated with thyroxine, which was subsequently withdrawn from three. Following thyroxine withdrawal, one infant resumed normal thyroid function and two developed compensated hypothyroidism. Of the five mothers, two had undiagnosed hypothyroidism and three were receiving thyroxine for longstanding hypothyroidism. Thyroid peroxidase antibody (aTPO) was measured in four and was negative in two, borderline positive in one and strongly positive in another. TRAb was measured in all five women and was strongly positive in all of them. This case series highlights the importance of identifying CH due to TRAb by investigating both the infant and the mother following a raised TSH found on newborn screening. The identification of those infants with transient CH caused by maternal transfer of TRAb is essential for optimizing management during childhood (including potential withdrawal of thyroxine replacement in the longer term) and in any subsequent pregnancy.

Fetal goitrous hypothyroidism due to maternal thyroid stimulation-blocking antibody: a case report.

Most fetal goitrous hypothyroidisms are reportedly caused by the maternal use of an antithyroid drug or fetal dyshormonogenesis. However, fetal goitrous hypothyroidism due to the transplacental passage of maternal thyroid stimulation-blocking antibody (TSBAb) is extremely rare. A woman at 28 weeks of gestation was found to have a fetal goiter by ultrasonography. Because the maternal serum showed hypothyroidism with an elevated titer of TSBAb, levothyroxine sodium was administered. The patient delivered a male infant, 3,412 g, with a goiter at term. Umbilical blood revealed primary hypothyroidism with increased TSBAb, and the infant was given levothyroxine sodium. After a month, neonatal thyroid function and TSBAb levels became normal. Attention should be paid to possible fetal hypothyroidism when a fetal goiter is observed to avoid impaired mental development of the neonate.

Detection of circulating autoantibodies against thyroid hormones in an infant with permanent congenital hypothyroidism and her twin with transient congenital hypothyroidism: possible contribution of thyroid hormone autoantibodies to neonatal and infant hypothyroidism.

Premature, very-low-birth-weight female twins were diagnosed with congenital hypothyroidism (CH). Thyroid hormone antibodies (THAb) directed against triiodothyronine (T3) and thyroxine (T4) of both the IgM and IgG class were detected in both twins. At age 10 months, a rise in thyroid stimulating hormone (TSH) occurred in both infants and measurement of THAb at the same time revealed the appearance of IgM-T4 and IgG-T4 and an increase in IgG-T3 levels in twin 1 (permanent CH), and with a slight increase of IgM-T4 and IgG-T4 levels and appearance of IgM-T3 in twin 2 (transient CH). THAb may contribute to the induction of hypothyroidism in neonates and infants, especially those with underlying autoimmune thyroid disease.

Thyroid peroxidase and thyroglobulin auto-antibodies in patients with newly diagnosed overt hypothyroidism.

OBJECTIVES: Thyroid autoimmunity is a major cause for hypothyroidism. We describe thyroid auto-antibodies in patients with various nosological subtypes of hypothyroidism identified in a population study. DESIGN: Population-based follow-up study identifying all new cases of hypothyroidism in an open cohort. METHODS: We established a monitoring system, and identified all new cases with primary overt hypothyroidism (n = 685) in a 4 year period in a well-defined population cohort (2,027,208 person-years of observation). Patients were sub-classified into: spontaneous hypothyroidism, presumably of autoimmune origin (n = 578); non-spontaneous hypothyroidism (associated with medication, delivery, neck-irradiation or subacute thyroiditis, n = 97); and congenital hypothyroidism (n = 10). A total of 186 adult patients (61% of those invited) underwent thyroid ultrasonography and measurements of antibodies against thyroid peroxidase (TPOAb) and thyroglobulin (TgAb). RESULTS: In spontaneously hypothyroid patients: >99% were antibody-positive (TPOAb or TgAb), TPOAb were more often measurable than TgAb (95.9 vs. 80.7%, p < 0.001). A statistically significant but modest correlation was observed between the two antibodies (Pearson's r2 = 0.11, p < 0.001). In a multivariate regression model both TPOAb and TgAb were positively associated with thyroid enlargement (p < 0.001), whereas no association was found with sex, age, iodine deficiency level or serum TSH level. We found no differences in patient characteristics between those who mainly developed TPOAb vs. those who preferentially harboured TgAb. CONCLUSIONS: Autoimmunity played a dominant role in practically all patients classified as spontaneously hypothyroid. Thyroid enlargement was associated with high levels of TPOAb and TgAb. We found no clue to why some spontaneously hypothyroid patients predominantly developed TPOAb whereas others mainly generated TgAb.

Benign thyroid disease: what is the role of nuclear medicine?

Nuclear medicine is directly involved in both the diagnosis and treatment of benign thyroid disease, which requires an understanding of the pathophysiology and management of thyroid disorders in addition to expertise in nuclear methodology. Thyroid uptake and imaging, the principal nuclear tests in thyroid disease, may be used as follows: (1) Differential diagnosis of hyperthyroidism: A very low thyroid uptake suggests destructive ("subacute") thyroiditis, a self-limited disorder, whereas a normal or elevated uptake is consistent with toxic nodular goiter and Graves' disease. Scintigraphic characteristics also help differentiate between nodular and Graves' disease. (2) Function of thyroid nodules: Fine-needle aspiration biopsy with cytological examination (FNAB) is used routinely to assess for malignancy in thyroid nodules. Scintigraphy may be of assistance before FNAB. "Hot" nodules are generally benign and do not require FNAB, while "cold" nodules may be malignant. (3) Differential diagnosis of congenital hypothyroidism: Scintigraphy combined with ultrasound examination may be used to identify such conditions as thyroid agenesis, dyshormonogenesis, and incomplete thyroid descent. Treatment of Graves' disease and toxic nodular disease with (131)I may require greater clinical involvement and decision analysis compared with thyroid uptake and imaging. The following aspects of treatment are particularly important: (1) Risk: Radioiodine treatment may occasionally aggravate hyperthyroidism, Graves' ophthalmopathy, and airway obstruction caused by large, nodular goiters. Alternative treatments, including the temporary use of antithyroid drugs, and surgery for nodular goiters, may be considered. (2) Radioiodine dose: Cure of hyperthyroidism with a single (131)I treatment is desirable, though not always possible. Such factors as a large goiter, severe hyperthyroidism, and prior propylthiouracil therapy, may contribute to treatment failure. (3) Informed consent: A detailed discussion with the patient regarding the clinical risks, outcomes, and side effects of (131)I is a critical component of successful management.

Humoral thyroid autoimmunity is not involved in the pathogenesis of myxedematous endemic cretinism.

A role of thyroid autoimmunity in the pathogenesis of myxedematous endemic cretinism was suggested by reports indicating the presence of thyroid growth-blocking antibodies in the sera of these patients. To check this hypothesis, we searched for TSH receptor antibodies with thyroid growth-blocking or adenylate cyclase (AC)-inhibiting (TSH-blocking) activity in immunoglobulin G (IgG) from 18 euthyroid and 21 hypothyroid endemic cretins living in Italy and Peru. Among hypothyroid cretins, 12 had no palpable goiter. Stages I-III goiters were present in 12 of 18 euthyroid cretins. Controls included 25 euthyroid nongoitrous subjects living in the same endemic regions as cretins, and 10 normal subjects from an iodine-sufficient area. IgG from 4 selected patients with autoimmune atrophic thyroiditis and from 2 neonates with sporadic transient congenital hypothyroidism due to maternal TSH-blocking antibodies were included in the study. The blocking effect of the IgG was assessed in FRTL-5 cells by measuring TSH-stimulated [3H]thymidine incorporation, DNA accumulation, and AC activation. A radioreceptor assay was used to detect TSH-binding inhibiting antibodies (TBIAb). No IgG from hypothyroid endemic cretins without goiter contained TBIAb or inhibited TSH-stimulated cell growth or AC activation. The effect of IgG from hypothyroid nongoitrous cretins did not differ from that produced by IgG from hypothyroid cretins with goiter, euthyroid cretins with or without goiter, or normal controls. In contrast to these results, IgG from patients with autoimmune atrophic thyroiditis and from neonates with sporadic transient congenital hypothyroidism contained TBIAb that inhibited both TSH-stimulated cell growth and AC activation. In conclusion, our results indicate that, similar to other types of endemic cretinism, hypothyroid endemic cretins without goiter do not have TSH receptor antibodies able to inhibit TSH-stimulated thyroid cell growth or function. These observations argue against a role of humoral thyroid autoimmunity in the development of myxedematous endemic cretinism.

Thyroid autoimmunity and neuropsychological development.

Thyroid autoimmunity may affect neuropsychological development by interfering with the fetal and/or neonatal thyroid status; indeed, both hypo- and hyperthyroidism during the fetal-neonatal life may be associated with a subsequent impairment of intellectual and psychomotor performances. Maternal thyroid status during early pregnancy, and in particular hypothyroidism due to autoimmune thyroiditis, can also affect the fetal neurological development. Finally, anti-thyroid drugs used to control hyperthyroidism in pregnant women with Graves' disease have been widely investigated for their possible effect on the intellectual development of the offspring.

Blocking type immunoglobulins in patients with nongoitrous primary hypothyroidism in area of iodine deficiency.

We have evaluated the role of circulating serum immunoglobulins (IgG) which inhibit the growth of thyroid in the etiology of thyroid atrophy in endemic cretinism. Twenty nongoitrous cretins (13 women and 7 men, age range: 9-33) were classified on the basis of clinical criteria for cretinism in China. They were born and living in an iodine deficient area, Xinjiang, northwest China. Antimicrosomal antibody titers were negative in all serum. Nine patients (seven women and two men; age range: 11-23) were biologically primary hypothyroid. Seven subjects were of a myxedematous form and two subjects were of a mixed form. We have studied thyroid-growth inhibiting immunoglobulin (TGII) activity that was measured as an inhibitory effect of 4 mg/ml IgG on TSH-induced [3H]-thymidine incorporation into the DNA of a rat thyroid follicular cell line, FRTL5 cells. Six (five women and one man) out of the nine patients with primary hypothyroidism (66.7 percent) had TGII. We also measured other growth-blocking IgG that inhibited [3H]-thymidine incorporation into DNA stimulated by insulin-like growth factor-I (IGF-I), a growth factor working through a cAMP-independent pathway. Five (three women and two men) out of nine patients (55.6 percent) with nongoitrous primary hypothyroidism had IGF-I-blocking IgG. These results indicate that TGII plays an important role in atrophy of the thyroid in spite of increased serum TSH concentrations, and IgG which inhibits thyroid growth stimulated by IGF-I also might play a role in thyroid atrophy in some endemic cretins.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid atrophy in myxedematous endemic cretinism: possible role for growth-blocking immunoglobulins.

We have examined the ability of IgGs obtained from 8 endemic cretins to inhibit TSH-stimulated thyroid cell growth in culture. Clinical and laboratory evidence for hypothyroidism was present in six subjects; the two remaining patients had borderline low serum T4, normal T3 and exaggerated TSH response to TRH. In six patients 2 mg IgG exhibited an inhibitory effect in the cellular growth expressed by a diminished incorporation of 3H-thymidine into the DNA of TSH-stimulated FRTL-5 cells (range: 26-87% inhibition). Seven patients presented clinically with thyroid atrophy of relatively small thyroid enlargements for the degree of chronic iodine deficiency that was present in the area. The remaining subject had a large multinodular goiter and IgG purified from this patient had no inhibitory effect in the FRTL-5 cellular growth. A direct relationship was noted between the degree of thyroid growth inhibition (%) and the basal serum TSH concentration. We conclude that the presence of thyroid growth inhibiting immunoglobulin may be related to the absence of thyroid growth or even thyroid atrophy in endemic cretins.

Endemic cretinism: possible role for thyroid autoimmunity.

Thyroid atrophy, rather than goitre, is a characteristic feature of myxoedematous cretinism but its cause and nature are unknown. In this study, purified IgG fractions of serum from patients with myxoedematous endemic cretinism inhibited thyrotropin-induced DNA synthesis in guineapig thyroid segments in a sensitive cytochemical bioassay. IgG from patients with euthyroid neurological endemic cretinism or from normal subjects did not inhibit thyroid growth. Furthermore, in myxoedematous subjects, the presence of the thyroid-growth-blocking immunoglobulins showed a positive relation with thyroid atrophy found on ultrasound. These findings provide a pathogenic basis for the variable clinical expression of endemic cretinism.

Autoimmune thyroid disease: an expanding spectrum.

Autoimmune thyroid disease classically has included Hashimoto's thyroiditis and Graves' disease. Hashimoto's thyroiditis probably also includes focal thyroiditis, fibrous thyroiditis, primary myxedema, and Hashitoxicosis as variants. Graves' disease is associated with ophthalmopathy and dermopathy, and recent evidence suggests that these manifestations are autoimmune phenomena as well. Other associated autoimmune disorders include idiopathic thrombocytopenic purpura and antigen-antibody complex nephritis. Nonthyroid endocrine autoimmune deficiency disorders also have been classified as part of the spectrum of thyroid autoimmune disease. With the recent recognition of the spectrum of autoimmune mechanisms and antibody types and methods to distinguish antibody functions or types, our understanding of postpartum and neonatal thyroid disorders has been advanced considerably. The spectrum of neonatal thyroid disorders in the infants of women with autoimmune disease relates to the levels and types of antithyroid antibodies acquired from the mother. Finally, there is suggestive evidence that nonspecific goiter, including simple adolescent goiter and multinodular goiter as well as some cases of sporadic cretinism, may be part of an even more expanded spectrum of autoimmune thyroid disease.