A rapid fluorometric enzyme immunoassay for the determination of neonatal TSH from blood spots.

We describe a novel method for the detection of thyrotropin from dried blood spots using a horseradish peroxidase-labelled sandwich enzyme immunoassay with fluorometric detection. The detection limit of the present assay is 1.25 mIU/l with within-run and between-run imprecision being in the range 5.2 to 11.4%. The results of the assay correlate well with two commercial methods: an enzyme immunoassay (r = 0.93) and a time-resolved fluorescence assay (r = 0.90). The blood spot values also show a good correlation (r = 0.93) with respective values obtained from plasma using a commercial immunoradiometric method. The assay may also be performed colorimetrically with sensitivity similar to the fluorometric assay. However, the latter provides a wider dynamic range with an upper limit of 400 mIU/l while the colorimetric method reaches a plateau at 25 mIU/l. Due to its simplicity and rapid performance (3 h), the fluorometric assay is suitable for the routine screening of congenital hypothyroidism.

3-p-hydroxyphenylpropionic acid--a sensitive fluorogenic substrate for automated fluorometric enzyme immunoassays.

The application of 3-p-hydroxyphenylpropionic acid (HPPA), a fluorogenic substrate of horseradish peroxidase (HRP) to an automated microplate fluorometric enzyme immunoassay is described. Fluorescence intensity of the end product was highly dependent on the pH of the buffer and on the concentrations of the substrate mixture ingredients. The determination of human thyrotropin (TSH) and recombinant hepatitis B surface antigen (rHBsAg) were performed using a fluorometric enzyme immunoassay (FEIA) with HPPA as the substrate, and a colorimetric one with tetramethylbenzidine (TMB) as the chromogenic substrate. The sensitivity of both types of assays proved comparable. The distinct advantage of a fluorometric assay is the possibility to perform a quantitative detection of analyte over a very wide dynamic range. Clinical evaluation of both assays showed good correlation between the FEIA and conventional methods.

Rapid determination of C-reactive protein by enzyme immunoassay using two monoclonal antibodies.

Several monoclonal antibodies for human C-reactive protein (CRP) were characterized, and two antibodies binding to separate domains were used to construct a rapid and simple immunoenzymometric assay for CRP. The assay consists of a single 15 min immunological reaction during which CRP forms a complex with a peroxidase-labelled antibody and with another antibody attached to the test-tube wall. The immobilized complex is detected by a 3 min colour reaction using peroxidase substrate. The quantitative measuring range of the assay is 0.04-5 mg/l, and no hook occurs at five-fold higher values. The sensitivity of the method allows reliable determination of low CRP levels, eg. in paediatric samples. The values obtained with the present assay correlated well with turbidimetric results.

Peripheral blood lymphocyte subsets in radiologists exposed to ionizing radiation.

To investigate whether low dose ionizing radiation caused perturbation of peripheral blood lymphocytes in radiology unit staff, the T-helper/suppressor ratio was examined in eight radiologists exposed to low dose radiation over a period of 6 to 27 years (mean 12 years). No significant difference was noted in the T-cell subsets between exposed radiologists and non-exposed control subjects. The effect of low dose ionizing radiation on peripheral blood lymphocyte subsets seems to be virtually negligible. Further, measurement of the T-helper/suppressor ratio is not a reliable way of demonstrating any damage to bone marrow caused by low dose ionizing radiation.

Lymphocyte subpopulations and reactivity during and after infectious mononucleosis.

T- and B-lymphocyte numbers, as well as lymphocyte reactivity to mitogens in vitro, were studied and correlated to other laboratory tests during the acute phase of infectious mononucleosis (30 patients) and up to one year thereafter. During the acute disease an absolute increase in both T- and B-lymphocyte numbers was recorded, the relative increase in B-lymphocytes occurring at the start. B-lymphocyte numbers fell after the second week and T-lymphocyte numbers after the fourth week of disease. Lymphocyte activation was impaired in all patients during the acute phase and was still significantly impaired for some mitogens after 6-9 months. Very few correlates between lymphocyte tests and other laboratory and clinical parameters were found.

In vitro production of leucocyte migration inhibitory factor by lymphocytes in exudative pleural effusions.

The indirect leucocyte migration inhibition test was used to investigate production of leucocyte migration inhibitory factor (LIF) by lymphocytes from pleural fluid and peripheral blood. The samples were taken from 19 patients with exudative pleural effusion resulting from malignant disease (n = 9), tuberculosis (n = 5) and nonspecific causes (n = 5). The lymphocytes were cultured in the presence of leucoagglutinin (LA) and purified protein derivative (PPD) and without mitogen or antigen. Although LIF production varied widely, pleural fluid lymphocytes from patients with benign diseases produced significantly more LIF in response to LA than pleural fluid lymphocytes from patients with malignant disease. In comparison to peripheral blood, the LA induced LIF production by pleural fluid lymphocytes was significantly enhanced in patients with nonspecific pleural effusion and significantly reduced in patients with malignant disease. However, pleural fluid lymphocytes from four patients with malignant disease produced LIF spontaneously, suggesting that they were already stimulated in vivo. In tuberculous pleural effusions the percentage of macrophages correlated negatively with the LIF production induced by LA and PPD. Lymphokines produced by sensitized lymphocytes in pleural fluid may be important in the local response to infectious agents and tumour growth by affecting the cellular composition of the effusion.

Leucocyte migration inhibition assay from clotted plasma droplets.

Two different techniques for performing leucocyte migration inhibition factor (LIF) assays were compared. A clotted plasma droplet LIF assay gives similar results as the conventional capillary tube method, but it is more sensitive and technically simpler to perform, and less blood is required for the assay. The plasma droplet method is suitable for particulate (BCG) as well as soluble (PPD) antigens, provided that the culture medium is supplemented with horse or human serum. If foetal calf serum is used no inhibition can be demonstrated with soluble antigen. Defibrinated blood gives the most clear-cut results and LIF activity can still be detected even after 24 hours storage of samples. EDTA anti-coagulated blood can alternatively be used, but it is less stable. The assay can still be performed after 3 hours storage of the blood, although the migration inhibition is decreasing. After 24 hours storage EDTA anticoagulated blood cannot be used for LIF assays.

Further decrease in thyroidal uptake and disappearance of endemic goitre in children after 30 years of iodine prophylaxis in the east of Finland.

Endemic goitre of moderate severity was mainly found in the east of Finland in the 1930's. Studies in the 1950's showed an average daily iodine intake of 65-70 micrograms in the west and 50-65 micrograms in the east of the country. The use of iodized salt was introduced in the late 1940's but added only 15 micrograms of iodine to the daily intake. In the late 1950's iodine prophylaxis was intensified and the use of salt containing 25 mg KI/kg was recommended. In 1978 about 95% of all household salt used in the Savonlinna area was iodized. This region in the east of Finland has been used as an area of surveillance and studies have been carried out there in 1959, 1969 and 1979. During this period the thyroidal uptake decreased from 67 to 23% in non-goitrous subjects and from 62 to 28% in goitre patients the difference between the two last figures being statistically significant. The goitre patients also had significantly higher serum thyroxine and triiodothyronine levels. During the same period the urinary excretion of stable 127I increased from 45 micrograms to about 250 micrograms a day. Concomitantly the goitre prevalence among school children has decreased. Having been in the early 1950's in most parts 15-30% it is generally now 1-4%. It seems that the iodine intake is now adequate and that the endemia is gradually subsiding.

Increase in thyroxine-binding globulin (TBG) in lysinuric protein intolerance.

The common occurrence of increased serum PBI concentration in patients with lysinuric protein intolerance (LPI) was elucidated by further studies. The reason was found to be an increase in the concentration of thyroid binding globulin (TBG), concomitantly with an increase in the binding capacity of TBG. The concentrations of serum thyroxine and triiodothyronine were elevated, whereas the free thyroxine index remained normal. The free triiodothyronine index was slightly increased. The binding capacity of thyroid hormone binding pre-albumin (TBPA) was significantly decreased. The concentrations of reverse triiodothyronine (3,3',5'-T3) and of 3,3'-diiodothyronine were normal. In all patients serum lactic acid dehydrogenase activities and ferritin concentrations were elevated. The reason for the almost constant increase in TBG remains obscure. It may be related to the primary disorder of LPI, a defect in diaminoacid transport.

Marathon run: effects on blood cortisol -- ACTH, iodothyronines -- TSH and vasopressin.

Blood cortisol, ACTH, thyroxine, triiodothyronine, reverse triiodothyronine, thyroid stimulating hormone (TSH) and vasopressin concentrations were determined in 9 runners (29-56 years old) and one 80 year old man taking part in a non-competitive Marathon in Athens, Greece on October 1976. After the run the mean concentrations of cortisol, ACTH and vasopressin showed a significant rise. The thyroid function variables and TSH did not differ from the control values. There was a significant correlation between the cortisol and ACTH levels after the race and also between their increments from the corresponding base values. A significant correlation was found between the physical fitness (as measured by indirect determination of VO2max) and the post-race cortisol levels. One of the well trained runners with a fairly good running time had the highest post-race values for 6 of 7 hormones studied.

Leucocyte migration inhibition factor (LIF) and antithrombin III (AT III).

The activity of LIF, produced in vitro by sensitized lymphocytes exposed to the appropriate antigens, is readily abolished in the simultaneous presence of heparin and thrombin. Heparin or thrombin alone are without significant effects. Adding AT III together with heparin and thrombin restores the LIF activity. We suggest that the migrating leucocytes on their cell membranes have receptors, composed of AT III-like molecules, which are blocked or destroyed, by forming complexes with heparin and thrombin. However, the heparin-thrombin complex will preferentially interact with AT III added to the culture medium, thus leaving the cellular receptors free to interact with LIF.

Thyroid hormones and TSH, prolactin and LH responses to repeated TRH and LRH injections in depressed patients.

Twelve patients with unipolar depressive disorders received 600 microgram of synthetic TRH or LRH, in a random order, for 3 days each. Placebo injections were given as two 3-day courses prior to and between the active treatments. Serum TSH, prolactin (Prl) and LH were measured by radioimmunoassays prior to the experiment as well as immediately before and 20 min after each injection. Serum T4 and T3 were determined by radioimmunoassays before the treatments and 24 h after the first two TRH injections. Serum T4 level in depressed patients did not differ from controls. Serum T3 level in depressed patients was significantly below, and the reverse T3 level was slightly above the normal mean. However, the latter difference was not statistically significant due to the large variation of rT3 levels among the depressed patients. The TSH responses did not differ from that of controls after the first injection but the responses after the second injection were lower than in a control study. The Prl responses to TRH were sub-normal in seven out of eight patients. Blunted LH responses to LRH were seen in three patients.