BL-22-CT : 100 determinations

The measurement of LH and FSH concentrations in serum and urine is essential for investigating fertility and especially disorders of the hypothalamic/ pituitary/gonadal axis. Both LH and FSH are secreted by the basophil cells of the anterior pituitary as a result of gonadotropin releasing hormone (GnRH) secretion from hypothalamic cells. In adults, LH and FSH hormones control gonadal functions; mainly gametogenesis and steroid secretion.

Circulating levels of FSH are controlled by a negative feedback effect on the hypothalamus by steroidal hormones and gonadal peptides.

The Bio-Line FSH-Irma is an immunoradiometnc assay based on coated-tube separation. Mabs1, 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-FSH tracer: 1 vial (red solution) containing 5.5 ml. Activity < 19µCi or 700 kBq.

3. Coated tubes: 2 x 50 tubes, coated with Anti-FSH 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, 100 µl, 500 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.

A. 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, heparinized or EDTA plasma provide similar results.

                    Y (serum) = 0.96x (hep. plasma) + 0.18 r = 0.99 n = 28

                    Y (serum) = 0.90x (EDTA plasma) + 0.28 r = 0.99 n = 28

B. Urine : Urine has to be diluted 4 times with zero standard.

If the assay is performed within 7 days, the urine shouid be kept at 2 to 8°C. Freezing must be avoided.

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

2. Vortex mix briefly standards, samples, controls and dispense 100 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 1 hour at room temperature on a tube shaker (700 rpm).

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 FSH (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.

                INTRA ASSAY                                                                                                 INTER ASSAY

C. Speciflcity

Cross-reactive hormones were added to a low FSH value serum and to a high value standard (50 mIU/ml). The apparent FSH response was measured.

D. Accuracy

                                                                                RECOVERY TEST

                                                                                DILUTION TEST

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Pulsatility of reproductive hormones: physiological basis and clinical implications.

Baillière’s Clin. Endocrinol. Metab., 1:1

2. FILICORI M., SANTORO N., MERRIAN G.R., CROWLEY W.F. Jr.,(1986)

Characterization of the physiological pattern of episodic gonadotropin secretion throughout the human menstrual cycle.

J. Clin. Endocrinol. Metab., 62:1136

3. MAIS V., CETEL N.S., MUSE K.N., QUIGLEY M.E., REID R.L., YEN S.S.C. (1987)

Hormonal dynamics during luteal-follicular transition.

Clin. Endocrinol Metab., 64:1109

4. SAWYER-STEFFAN J.E., LASLEY B.L., HOFF J.D., YEN S.S.C. (1982)

Comparison of in-vitro bioactivity and immunoreactivity of serum LH in normal cyclic and hypogonadal women treated with low doses of LH-RH.

J. Reprod. Fertil., 65:45

5. THOMAS C.M.G., SEGERS M.F.G. (1988)

Measurement of Serum hLH: hCG interference evaluated for two hLH-Speciflc IRMA kits.

Clin. Chem., 34:768

6. LOUMAYE E., VANKRIEKEN L., DEPREESTER S., PSALTI I., de COOMAN S., THOMAS K. (1989)

Hormonal changes induced by short-term administration of a gonadotropin-releasing hormone agonist during ovarian hyperstimulation for in vitro fertilization and their consequences for embryo development.

Fertil. and Steril., 51:105

7. VERMES I., BONTE HA., SLUIS VEER G., SCROEMAKERJ. (1991)

Interpretations of five monoclonal immunoassays of Lutropin and Follitropin: effects of normalization with WHO standard.

Clin. Chem., 37:415

8. DEMOULIN A., DUBOIS M., GERDAY C., GILLAIN D., LAMBOTFE R., FRANCHIMONT P. (1991)

Variation of luteinizing hormone serum concentration after exogeneous human chorionic gonadotropin administration during ovarian stimulation.

Fertil. and Steril., 55:796

9. MASSA G., de ZECHER F., VANDERSCHUREN.LODEWYK K. (1992)

Serum levels of immunoreactive Inhibine, FSH and LH in human infants at Preterm and Term Birth.

Biol. Of the Neonat, 61:150

10. DE HERTOGH R., VANKRIEKEN L., THOMAS K. de GASPARO M. (1992)

Circhoral fluctuations of serum total renin, inhibin and related hormones around the mid-cycle in normal human females.

Hum. Reprod., 7:337