BL-13-CT

Thyrotrope cells of the anterior pituitary synthesize and secrete human thyroid stimulating hormone (TSH), a glycoprotein of molecular weight 28,000, comprising two subunits :α-TSH is very similar to a α subunit of LH, FSH and hCG, β-TSH differs from other hormone subunits and defines the immunological specifity.

TSH regulates the synthesis and release of thyroid hormones : thyroxine (T4) and triiodothyronine (T3). TSH secretion is stimulated by an hypothalamic peptide, TRH (TSH releasing hormone) ; a negative feedback on TSH secretion is exerted by T3 and T4. Primary hyperthyroidism is now easily differentiated from euthyroidism by Bio-Line ultrasensitive TSH-Irma, because of the high sensitivity (0.025 µIU/mL) and high discrimation power.

The Bio-Line TSH-Irrna is an immunoradiometnc assay based on coated-tube separation. Mabs 1, the capture antibodies, are attached to the lower and inner surface of the plastic tube. Standards or samples added to the tubes will at first show low affinity for Mabs1. Addition of Mab2, the signal antibody labelled with ¹²⁵I, will complete the system and trigger the immunological reaction. After washing, the remaining radioactivity bound to the tube reflects the antigen concentration. The use of several distinct Mabs avoids hyperspecificity.

1. Radioactive material: Radioactive material may be received, acquired, possessed and used only by                     physicians, clinical laboratories, or hospitals for "In-Vitro" clinical or laboratory tests not involving                                    internal or external administration of the material, or the radiation therefrom, to human beings or animals.

Compliance with these basic rules of radiation safety should provide adequate protection:

1. Do not eat, drink, smoke, or apply cosmetics in areas where radioactive material is used.

2. Do not pipet by mouth reagents containing radioactive materials.

3. Wear protective clothing; i.e., lab coats and disposable gloves, in order to avoid direct contact with radioactive reagents.

4. Work with radioactive materials should be performed in a designed area.

5. Radioactive materials should be stored in an acceptable location.

6. A log should be kept for receipt and disposal of radioactive materials.

7. Radioactive spills or accidents should be taken care of immediately according to established procedures.

8. Disposal of radioactive materials must comply with prevailing regulations and guidelines of the agencies holding jurisdiction over the laboratory.

 

2. Sodium azide: Sodium Azide, used as a bacteriostatic agent, is toxic in acid medium. In addition, it may form                 potentially explosive lead or copper azides. To avoid dangerous deposits, waste solutions should be flushed                 away with large volumes of water.

3. Hepatitis and Acquired Immune Deficiency Syndrome (HTLV-III): All Bio-Line reagents included in this                 kit have been tested and found to be non reactive for hepatitis B surface antigen. They have also been                              screened and determined to be non-reactive for HTLV-III antibody. However, human serum products should be                 handled as if potentially capable of transmitting hepatitis, Acquired Immune Deficiency Syndrome, or other                 infectious agents.

Kit contains sufficient reagents for 100 determinations.

2. ¹²⁵I TSH tracer: 1 vial (red solution) containing 5.5 ml. Activity < 19µCi or 700 kBq.

3. Coated tubes: 2 x 50 tubes, coated with Anti-TSH monoclonal antibodies.

4. Wash solution concentrate: 1 vial of 10 ml of concentrate, to be diluted into 700 ml distilled water and stored at 4° C.

The following material is required but not provided in the kit:

1. Distilled water

2. Pipettes for delivery of: 50 µl, 200 µl, 1 ml and 2 ml (the use of accurate pipettes with disposable plastic tips is recommended)

3. 5 ml automatic syringe (Cornwall type) for washing

4. Aspiration system (optional)

5. Gamma counter, set for ¹²⁵I counting and Vortex mixer and magnetic stirrer.

Serum or plasma : Serum and plasma must be kept at 2 – 8°C.

If the test is not run within 24 hours, storage at –20°C is recommended and will not result in loss of immuno-reactivity for at least 6 months.

Serum or EDTA plasma provide similar results.

            Y (serum) = 1.05x (hep. plasma) + 0.03 r = 0.97 n = 30

            Y (serum) = 1.00x (EDTA plasma) + 0.03 r = 0.98 n = 30

1. Label coated tubes in duplicate for each standard, sample, control. For determination of total counts, label 2 normal tubes.

2. Vortex briefly standards, samples, controls and dispense 200 µl of each into the respective tubes.

            3. Dispense 50 µl of tracer into each tube.

4. Shake the rack containing the tubes gently by hand.

5. Incubate for 2 hours at room temperature on a shaker.

6. Aspirate (or decant) the content of each tube (except total counts). Be sure that the plastic tip of the aspirator reaches the bottom of the coated tube in order to remove all the liquid.

7. Wash the tubes with 2 ml Wash Solution (except total counts). Avoid foaming during the addition of the Wash Solution.

8. Aspirate (or decant) the content of each tube (except total counts).

9. Wash again tubes with 2 ml Wash Solution (except total counts) and aspirate (or decant).

10. After the last washing let the tubes standing upright for 2 minutes and aspirate the remaining drop of liquid.

11. Count the tubes in a gamma counter for 60 seconds.

On semilogarithmic or linear graph paper plot the c.p.m. (ordinale) for each standard against the corresponding concentration of TSH (abscissa) and draw a standard curve through the standard points, reject the obvious outliers. Read the concentration for each control and sample by interpolation on the standard curve. Computer assisted data reduction will simplify these calculations.

            B. Precision

            INTRA ASSAY                                                                            INTER ASSAY

DILUTION TEST

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2. CALDWELL G., KELLET H.A. , GOW S.M., et al.A new strategy for thyroid function testing.Lancet 1985, I :1117-9

3.FIELD J.B. In the thyroid, S. Werner and S.H. Ingbar. Ed. Harper Row, Hagestown, M.D..,1978.

4. MARDWELL R.T., GAMBER T.R., WINTON M.R.J.High sensitivity assay of thyroid stimulating hormone in patients receive thyroxine for primary hypothyroidism and thyroid carcinoma.BR. Med. J. 1985 : 290 :335-356.

5. MUSTO J.D., PIZZOLANTE J.M. and CHESARONE V.P.A comment on thyrtropin : Chemistry.Clin. Chem., 1984 ;30 :329

6. PIERCE J.C.Pituitary thyrotropin : Chemistry.In the thyroid., S. Werner and S.H. Ingbar. ED. Harper Row, Hagerstown, M.D.1978.

7. RODDIS M.J., BURRIN J.M., JOHANNSSEN A., et al.Serum thyrotropin : a first-line discriminatory test of thyroid function.Lancet 1985 ; I :277-8

8. ROSS D.S.New sensitive immunoradiometric assays for thyrotropin (Review).Ann. Intern. Med. 1986 ; 104 ;718-21

9. PETER S.A. et al.Elevated serum thyrotropin (TSH) levels in critically ill patients with acquired immunodeficiency syndrome (AIDS).Exp. Clin. Endocrinol. 1993 ; 101(6) ;346-9

10. JAIMELA E. et al.Ability of two new thyrotropin (YSH) assays to separate hyperthyroid patients from euthyroid patients with ow TSH.Clin. Chem. 1994, Jan ;40(1) ;101-105

11. KOMOROWSKI J. et al.Stimulatory effect of thyroprotin (TSH) on interleukin 2 (IL2) release from human peripheral bllod lymphocytes and dose-response study in vitro.Horm. Metab. Res. 1993,Nov.;25(11).;598-9.