3.      Principles of the method

A fixed amount of ¹²⁵I labelled Androstenedione competes with the Androstenedione to be measured present in the sample or in the calibrator for a fixed amount of antibody sites being immobilized to the wall of a polystyrene tube. After 1 hour incubation at room temperature on a shaker, an aspiration step terminates the competition reaction. The tubes are then washed with 2 ml of Working Wash Solution and aspirated.  A calibration curve is plotted and the Androstenedione concentrations of the samples are determined by dose interpolation from the calibration curve.

4.       Reagents provided

Note :      Use the zero calibrator for sample dilutions.

5.      Supplies not provided

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

1.           Distilled water

2.           Pipettes for delivery of: 25 µl, 250 μl, 500 µl and 2 ml (the use of accurate pipettes                 with disposable plastic tips is recommended)

3.           Vortex mixer

4.           Magnetic stirrer

5.           Tubes shaker (700 rpm)

6.           5 ml automatic syringe (Cornwall type) for washing

7.           Aspiration system (optional)

8.           Any gamma counter capable of measuring ¹²⁵I may be used (minimal yield 70%).

6.     Reagent preparation

A.       Calibrators : Reconstitute the calibrators with 0.5 ml distilled water

B.       Controls: Reconstitute the controls with 0.5 ml distilled water.

C.       Working Wash solution: Prepare an adequate volume of Working Wash solution by adding 69 volumes of distilled water to 1 volume of Wash Solution (70x). Use a magnetic stirrer to homogenize. Discard unused Working Wash solution at the end of the day.

7.      Storage and expiration dating of reagents

-         Before opening or reconstitution, all kit components are stable until the expiry date, indicated on the label, if kept at 2 to 8°C.

-         After reconstitution, calibrators and controls are stable for one week at 2 to 8°C. For longer storage periods, aliquots should be made and kept at ‑20°C for maximum 3 months.  Avoid successive freezing and thawing.

-         Freshly prepared Working Wash solution should be used on the same day.

-         After its first use, the tracer is stable until expiry date, if kept in the original well-closed vial at 2 to 8°C.

-         Alterations in physical appearance of kit reagents may indicate instability or deterioration.

8.      Specimen collection and preparation

-         Serum or plasma samples must be kept at 2‑8°C.

-         If the test is not run within 24 hrs, storage in aliquots at ‑20°C is recommended.

-         Avoid successive freezing and thawing.

-         Serum and plasma provides similar results:

                Y (Hep. plasma) = 0.94 x (serum) + 0.02               r = 0.98    n = 17

                Y (EDTA plasma) = 1.01 x (serum) – 0.03r = 0.95     n = 17

9.       Procedure

A.       Handling notes

          Do not use the kit or components beyond expiry date.  Do not mix materials from different kit lots.  Bring all the reagents to room temperature prior to use.

          Thoroughly mix all reagents and samples by gentle agitation or swirling.

          In order to avoid cross-contamination, use a clean disposable pipette tip for the addition of each reagent and sample.

          High precision pipettes or automated pipetting equipment will improve the precision.  Respect the incubation times. 

          Prepare a calibration curve for each run, do not use data from previous runs.

B.       Procedure

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

2.           Briefly vortex calibrators, controls and samples and dispense 25 μl of each into respective tubes.

3.           Dispense 250 µl of ¹²⁵Iodine labelled Androstenedione into each tube, including the uncoated tubes for total counts.

4.           Shake the tube rack gently by hand to liberate any trapped air bubbles.

5.           Incubate for 1 hour at room temperature with continuous shaking.

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 tubes with 2 ml Working Wash solution (except total counts) and aspirate (or decant). Avoid foaming during the addition of the Working Wash solution.

8.           Let the tubes stand upright for two minutes and aspirate the remaining drop of liquid.

9.           Count tubes in a gamma counter for 60 seconds.

10.      Calculation of results

1.        Calculate the mean of duplicate determinations.

2.        Calculate the bound radioactivity as a percentage of the binding determined at the zero calibrator point (0) according to the following formula :

4.        Computer assisted methods can also be used to construct the calibration curve. If automatic result processing is used, a 4-parameter logistic function curve fitting is recommended.

5.        By interpolation of the sample (B/B0 (%)) values, determine the Androstenedione concentrations of the samples from the calibration curve.

6.        For each assay, the percentage of total tracer bound in the absence of unlabelled Androstenedione (B0/T) must be checked.

11.     Typical data

The following data are for illustration only and should never be used instead of the real time calibration curve.

12.    Performance and limitations

A.       Detection limit

          Twenty zero calibrators were assayed along with a set of other calibrators.

          The detection limit, defined as the apparent concentration two standard deviations below the average counts at zero binding, was  0.03 ng/ml.

B.       Specificity

The specificity of the polyclonal antibody used in this assay was evaluated by RIA, as the ratio of the amount of Androstenedione which yields 50% inhibition of binding of labelled Androstenedione and the amount of cross-reacting compounds (analogs) giving the same inhibition.

Note :      This table shows the cross-reactivity for the anti Androstenedione.

          ND : not detectable

C.      Precision

SD: Standard Deviation; CV: Coefficient of variation

D.       Accuracy    

DILUTION  TEST

Samples were diluted with the zero calibrator.

RECOVERY  TEST

To the best of our knowledge, no international reference material exists for this parameter.

E.       Time delay between last calibrator and sample dispensing

          As shown hereafter, assay results remain accurate even when a sample is dispensed 24 minutes after the calibrator has been added to coated tubes.

TIME DELAY

13.   INTERNAL  QUALITY  CONTROL

-         If the results obtained for Control 1 and/or Control 2 are not within the range specified on the vial label, the results cannot be used unless a satisfactory explanation for the discrepancy has been given.

-         If desirable, each laboratory can make its own pools of control samples, which should be kept frozen in aliquots.

-         Acceptance criteria for the difference between the duplicate results of the samples should rely on Good Laboratory Practises.

14.       REFERENCE  INTERVALS

These values are given only for guidance; each laboratory should establish its own normal range of values.

The ranges are expressed as 2.5% to 97.5% percentiles.

15.   PRECAUTIONS  AND  WARNINGS

Safety

For in vitro diagnostic use only.

This radioactive product can be transferred to and used only by authorized persons; purchase, storage, use and exchange of radioactive products are subject to the legislation of the end user's country.  In no case the product must be administered to humans or animals.

All radioactive handling should be executed in a designated area, away from regular passage.  A logbook for receipt and storage of radioactive materials must be kept in the lab.  Laboratory equipment and glassware, which could be contaminated with radioactive substances, should be segregated to prevent cross contamination of different radioisotopes.

Any radioactive spills must be cleaned immediately in accordance with the radiation safety procedures.  The radioactive waste must be disposed of following the local regulations and guidelines of the authorities holding jurisdiction over the laboratory.  Adherence to the basic rules of radiation safety provides adequate protection.

The human blood components included in this kit have been tested by European approved and/or FDA approved methods and found negative for HBsAg, anti-HCV, anti-HIV-1 and 2.  No known method can offer complete assurance that human blood derivatives will not transmit hepatitis, AIDS or other infections.  Therefore, handling of reagents, serum or plasma specimens should be in accordance with local safety procedures.

All animal products and derivatives have been collected from healthy animals. Bovine components originate from countries where BSE has not been reported. Nevertheless, components containing animal substances should be treated as potentially infectious.

Avoid any skin contact with reagents (sodium azide as preservative).  Azide in this kit may react with lead and copper in the plumbing and in this way form highly explosive metal azides.  During the washing step, flush the drain with a large amount of water to prevent azide build-up.

Do not smoke, drink, eat or apply cosmetics in the working area.  Do not pipette by mouth.  Use protective clothing and disposable gloves. 

16.  BIBLIOGRAPHY

1.        Dorfman RI, Shipley RA : Androgens. John Wiley and Sons, New York, pp. 116-128, 1956.

2.        Horton R, Tait J : Androstenedione production and interconversion rates measured in peripheral blood and studies on the possible site of its conversion to testosterone. J Endocrinol Invest 45:301-313,1966.

3.        Pang S, Riddick L : Hirsutism. IN Lifshitz T (ed) : Pediatric Endocrinology, A Clinical Guide, second edition. Marcel Dekker, Incl., New York, pp. 259-291, 1990.

4.        Cavallo A, Corn C, Bryan GT, Meyer WJ III : The use of plasma androstenedione in monitoring therapy of patients with congenital adrenal hyperplasia. J Pediatr 95:33-37, 1979. Bull NY Acad Med 53, 347, 1977

5.        Barett-Connor E, Garland C, McPhililips JB, Kaw K-T, Wingard DL :  A prospective, population based study of androstenedione, estrogens and prostate cancer. Canc res 50:169-173, 1990.

6.        Rittmaster RS, Thompson DL : Effects of leuprolide and esametehasone o hair growth and hormone levels in hirsute women : the relative importance of the ovary and adrenal in the pathogenesis of hirsutism. J Clin Endocrinol Metab 70:112-116, 1993.

7.        Zwicker H, Rittmaster RS : Androsterone sulfate : Physiology and signifiance in hirsute women. J Clin Endocrinol Metab 76:112-116, 1993.

17.       SUMMARY OF THE PROTOCOL