RADIOIMMUNOASSAY FOR THE DIRECT DETERMINATION OF 17 BETA-ESTRADIOL IN HUMAN SERUM OR PLASMA

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1 EstradCE.fm Page 1 Friday, September 19, :49 PM RADIOIMMUNOASSAY FOR THE DIRECT DETERMINATION OF 17 BETA-ESTRADIOL IN HUMAN SERUM OR PLASMA 1. INTRODUCTION 17 beta-estradiol is a female steroid hormone with a molecular weight of approximately 272 daltons, which is secreted by the ovaries. The primary function of estradiol is to prepare the uterine mucosa for the pregestational stage. It also suppresses the production of follicle-stimulating hormone (FSH) and stimulates preovulatory luteinizing hormone (LH) release from the pituitary (1). In serum estradiol is complexed to a binding protein (SHBG = sex hormone binding globulin) (5, 6). As in the case of other small molecular weight hormones, this binding protein may be involved in the regulation of hormone delivery to target organs (7). In non-pregnant women, estradiol serum levels are cyclic with peak levels occuring shortly before ovulation (2). Estradiol concentrations are useful in evaluating a variety of menstrual dysfunctions, feminization in children and estrogen-producing tumours. Estradiol may also be elevated in gynecomastia and cirrhosis. It is often measured in therapies aimed at overcoming infertility such as stimulation of ovulation by clomiphene or in vitro fertilization procedures (IVF) (3). Serum estradiol levels rise to very high concentrations during pregnancy. Therefore, monitoring of estradiol levels provides information relevant to fetal growth (4). In normal males, estradiol levels are very low. Abnormally elevated levels may be indicative of testicular tumours (1). 2. INTENDED USE The reagents in the kit are for in vitro diagnostic use only. This kit is to be used for the direct quantitative determination of 17 beta-estradiol levels in human serum and plasma. Range and performance of the method and its variations have been optimized to permit determinations of both very low (e.g. in children) and high (e.g. in IVF) concentrations of estradiol. 3. PRINCIPLE OF THE ASSAY This procedure for the direct measurement of estradiol is based on the competitive binding principle of radioimmunoassay. The samples and calibrators are incubated with estradiol tracer and antibody. After a second incubation followed by a centrifugation, separation of free tracer from antibody-bound tracer is achieved by means of a second antibody. After centrifugation, the tubes are decanted and the supernatants are discarded. The pellet is counted in a gamma counter. 1

2 EstradCE.fm Page 2 Friday, September 19, :49 PM A calibration curve is then prepared from serum calibrators ranging from 10 to 2000 pg/ml. Unknown values are obtained from the calibration curve by interpolation. 4. DESCRIPTION AND PREPARATION OF REAGENTS STORAGE: Upon receipt, the kit should be stored at 2-8 C. Do not freeze. Once opened, the reagents of this kit are stable until the kit expiry date when properly stored. The kit has been designed to perform 4 assay runs when used throughout the day at room temperature and stored overnight at 2-8 C. Reagents should not be used past the expiry date. The expiry date of the kit is reported on the external label and corresponds to the expiry date of the tracer. The expiry date of each component is reported on the respective vial label. When reconstituting the contents of the vials, mix gently to avoid foaming. Reagents from different batches must not be mixed I-tracer (ready-to-use reagent) The vial contains 10.5 ml phosphate buffer solution containing estradiol-6cmo- 125 I-iodohistamine, animal proteins, preservatives and an inert red dye. Radioactivity is 74 kbq (2 µci) or less on the calibration date Anti-estradiol antiserum (ready-to-use reagent) The vial contains 10.5 ml phosphate buffer solution containing antiserum raised in rabbits, animal proteins, preservatives and an inert blue dye Zero calibrator (ready-to-use reagent) The vial contains 1.25 ml human steroid-free serum, and preservatives Estradiol calibrators (ready-to-use reagent) Each vial contains 0.5 ml human steroid-free serum, preservatives and estradiol added to yield the concentrations reported below. The kit calibrators demonstrate commutability with patient samples when used with reagents and operating procedure of this in vitro diagnostic test as the manufacturer recommends. As no international standard preparation is currently available, the kit calibrators are calibrated against an internal reference preparation (99% purity by HPLC). 10 pg/ml = 37 pmol/l (Conversion factor: 1 pg/ml = 3.67 pmol/l) 40 pg/ml = 147 pmol/l 100 pg/ml = 367 pmol/l 400 pg/ml = 1468 pmol/l 1000 pg/ml = 3670 pmol/l 2000 pg/ml = 7340 pmol/l. 2

3 EstradCE.fm Page 3 Friday, September 19, :49 PM 4.5. Control serum (lyophilized reagent) The vial contains estradiol in human serum. The range of reference values is reported on the vial label. Reconstitute the vial contents with 1 ml distilled water. Store the resulting solution for two weeks at 2-8 C or in deep-frozen aliquots ( 20 C or below) for extended storage Precipitating reagent (ready-to-use reagent) The bottle contains 52 ml buffer, antibody to rabbit IgG raised in goats, non-specific rabbit IgG, polyethylene glycol and preservatives. Let the bottle of precipitating reagent reach room temperature and mix well by repeatedly tilting end over end. 5. EQUIPMENT AND SUPPLIES REQUIRED, BUT NOT PROVIDED Distilled and deionized water. 12 x 75 mm polystyrene or polypropylene test tubes. Test tube rack. Micropipettes with disposable tips (50, 100, 500 µl) (50 µl: trueness ± 3%, precision 2%; 100, 500 µl: trueness ± 2%, precision 1%). Calibrated pipettes (9, 10 ml). Graduated cylinders (50, 100 ml). Vortex mixer. Thermostatically-controlled water bath capable of maintaining 37 ± 1 C. Multisample centrifuge capable of achieving g. Gamma counter suitable for counting 125 I (counter window setting: kev - counter efficiency: 70% - counting time: 1 min). If counter efficiency is below 60%, counting time should be prolonged to 2 min. 6. ASSAY PREPARATION General sample preparation. Erroneous results can be caused by improper handling of patient samples. Either human serum or plasma may be used. The anticoagulants citrate, EDTA and heparin have been tested and may be used with this assay. Blood should be collected aseptically by venipuncture, allowed to clot, and the serum separated from the clot as soon as possible. Samples having particulate matter, turbidity, lipaemia, or erythrocyte debris may require clarification by filtration or centrifugation before testing. Grossly haemolyzed or lipaemic samples as well as samples containing particulate matter or exhibiting obvious microbial contamination should not be tested. If the assay is performed within 24 hours of sample collection, the samples should be kept at 2-8 C; otherwise they should be aliquoted and stored deep-frozen ( 20 C or below). If samples are stored frozen, thaw frozen samples at room temperature and mix gently before pipetting. Avoid 3

4 EstradCE.fm Page 4 Friday, September 19, :49 PM freeze-thaw cycles or foaming of patient samples. Improper sample storage may result in decreased trueness. Estradiol concentration can vary widely depending on the origin of the sample. Care should be taken that the expected concentration corresponds to the range of maximum precision of the assay. To optimize performance of the system the following guidelines may be used: Normal women (pre- and postmenopausal), men and children: No sample preparation necessary; use routine procedure (see section 7). For samples from women undergoing IVF treatment there are several alternatives: use special IVF procedures (see sections 8, 9), or dilute samples 1:2 with zero calibrator (to avoid loss of material, use 25 µl sample and directly dilute with 25 µl zero calibrator in the assay tube), and use routine procedure (see section 7). Sera of pregnant women should be diluted with steroid-free serum (PER20, supplied upon request) as reported herebelow. Trimester of pregnancy Patient serum (µl) Steroid-free serum (µl) Volume used per assay tube (µl) Dilution factor I II III To get the actual serum estradiol concentration, multiply result read off the calibration curve with appropriate dilution factor. Assay preparation. Assay reagents must be stored at 2-8 C and should be mixed thoroughly without foaming prior to use. All reagents must be brought to room temperature prior to use. The temperature in the water bath must be standardized uniformly and kept constant at 37 ± 1 C. The water level must be kept above that of the solution in the tubes without allowing the tubes to float. Controls should be included in every run. Failure to obtain the appropriate values for controls may indicate imprecise manipulations, improper handling or deterioration of reagents. Calibrators must be run with each series of patient specimens. Calibrators and samples should be subjected to the same process and incubation time. Perform all assay steps in the order given and without any appreciable delays between the steps. A clean, disposable tip should be used for dispensing each calibrator and sample. 4

5 EstradCE.fm Page 5 Friday, September 19, :49 PM Carefully aspirate the incubation mixture; failure to remove solution adequately may result in poor reproducibility and spurious results. No trace of dye should still be visible. 7. ROUTINE ASSAY PROCEDURE This procedure should be used when high precision and sensitivity of estradiol measurements are required (e.g. no samples from IVF patients). If a mixed population of samples, including IVF sera, is to be assayed, the procedure outlined in section 8 is recommended. The use of the 2000 pg/ml calibrator is optional in this procedure. Due to flatness of the calibration curve in the range from 1000 to 2000 pg/ml, precision may be poor despite the additional calibrator. Pipetting of total activity, non-specific binding, calibrators and unknowns is performed as reported below. Determinations should be done in duplicate. reagents tubes Total activity Zero calibrator (Bo) Calibrators 1-5 Samples Calibrators 50 µl 50 µl Samples 50 µl Tracer 100 µl 100 µl 100 µl 100 µl Antiserum 100 µl 100 µl 100 µl Shake tubes briefly (Vortex) and incubate for 2 hours in a 37 C water bath. Add 500 µl precipitating reagent to each tube except total activity. Shake tubes and incubate for 15 min at room temperature. After incubation, centrifuge the tubes at g for 15 min. Immediately after centrifugation, aspirate or decant the supernatant. Count the tubes for one minute. 5

6 EstradCE.fm Page 6 Friday, September 19, :49 PM Scheme of routine assay 50 µl calibrator/sample 100 µl tracer (red) 100 µl antiserum (blue) Incubate for 2 hours at 37 C in a water bath Add 500 µl precipitating reagent Incubate for 15 min at room temperature Centrifuge for 15 min at g Discard supernatant Count tubes for one min 8. PROCEDURE FOR SAMPLES OF IVF PATIENTS In this procedure all six calibrators are used. Together with the reduced sample volume a calibration curve results, that permits precision measurements in both the high and low concentration range. A sample volume of 25 µl is recommended here. However, even smaller volumes (20, 15 µl) can be used if necessary. Pipetting of total activity, non-specific binding, calibrators and unknowns is performed as reported in the following page. Determinations should be done in duplicate. 6

7 EstradCE.fm Page 7 Friday, September 19, :49 PM tubes Total Zero Calibrators calibrator activity 1-6 reagents (Bo) Samples Calibrators 25 µl 25 µl Samples 25 µl Tracer 100 µl 100 µl 100 µl 100 µl Antiserum 100 µl 100 µl 100 µl Shake tubes briefly (Vortex) and incubate for 2 hours in a 37 C water bath. Add 500 µl precipitating reagent to each tube except total activity. Shake tubes and incubate for 15 min at room temperature. After incubation, centrifuge the tubes at g for 15 min. Immediately after centrifugation, aspirate or decant the supernatant. Count the tubes for one minute. Assay scheme (25 µl sample) 25 µl calibrator/sample 100 µl tracer (red) 100 µl antiserum (blue) Incubate for 2 hours at 37 C in a water bath Add 500 µl precipitating reagent Incubate for 15 min at room temperature Centrifuge for 15 min at g Discard supernatant Count tubes for one min 7

8 EstradCE.fm Page 8 Friday, September 19, :49 PM 9. RAPID, ONE-INCUBATION PROCEDURE In this system, the first and second incubations are done in parallel. This results in reduced total assay time. The use of the 2000 pg/ml calibrator is optional. Pipetting of total activity, non-specific binding, calibrators and unknowns is performed as reported below. Determinations should be done in duplicate. Do not add antiserum. Premix antiserum and precipitating reagent in the proportions of 1:5 for the appropriate number of tubes. Example: number of tubes in assay: 30 antiserum required: 30 x 0.1 ml = 3 ml precipitating reagent to be added to antiserum: 5 x 3 ml = 15 ml. Stir mixture for 30 sec, immediately pipet 600 µl of mixture per tube. Premixed antiserum should be used within 15 min. The remainder of undiluted antiserum or precipitating reagent can be used in an assay according to section 7 or 8, or it can be stored according to section 4. reagents tubes Total activity Zero calibrator (Bo) Calibrators 1-5 Samples Calibrators 25 µl 25 µl Samples 25 µl Tracer 100 µl 100 µl 100 µl 100 µl Premixed antiserum/precipitating reagent 600 µl 600 µl 600 µl Shake tubes briefly (Vortex) and incubate for 2 hours in a 37 C water bath. After incubation, centrifuge the tubes at g for 15 min. Immediately after centrifugation, aspirate or decant the supernatant. Count the tubes for one minute. 8

9 EstradCE.fm Page 9 Friday, September 19, :49 PM Scheme of assay with premixed reagents 25 µl calibrator/sample 100 µl tracer (red) Mix one part of antiserum with 5 parts of precipitating reagent Add 600 µl premixed antiserum and precipitating reagent Incubate for 2 hours at 37 C in a water bath Centrifuge for 15 min at g Discard supernatant Count tubes for one min 10. CALCULATION OF RESULTS Many gamma counters now have automatic data reduction facilities. Where this is not available, it is recommended that the following procedure be followed. Compute the mean net counts for each determination in duplicate. Compute maximum binding (Bmax): Bmax% = zero calibrator mean counts total activity mean counts x 100 9

10 EstradCE.fm Page 10 Friday, September 19, :49 PM Compute the B/Bo ratio for each calibrator and unknown sample as follows: Plot in semilog or log-log coordinates the mean percent value for each calibrator on the ordinate (y axis) as a function of estradiol concentration expressed as pg/ml on the abscissa (x axis). A calibration curve is thus obtained (Fig. 1). Directly from the calibration curve, read the estradiol concentration of each sample expressed as pg/ml. If the sample was diluted, the estradiol concentration value derived from the diluted specimen must be multiplied by the dilution factor. Typical data (routine assay, see section 7). Typical data (25 µl sample, see section 8). 10 B/Bo% = calibrator or sample mean counts x 100 zero calibrator mean counts Description Concentration cpm % B/Bo Total activity (T) Zero calibrator (Bo) 0 pg/ml (0 pmol/l) Calibrator 1 10 pg/ml (37 pmol/l) Calibrator 2 40 pg/ml (147 pmol/l) Calibrator pg/ml (367 pmol/l) Calibrator pg/ml (1468 pmol/l) Calibrator pg/ml (3670 pmol/l) Description Concentration cpm % B/Bo Total activity (T) Zero calibrator (Bo) 0 pg/ml (0 pmol/l) Calibrator 1 10 pg/ml (37 pmol/l) Calibrator 2 40 pg/ml (147 pmol/l) Calibrator pg/ml (367 pmol/l) Calibrator pg/ml (1468 pmol/l) Calibrator pg/ml (3670 pmol/l) Calibrator pg/ml (7340 pmol/l)

11 EstradCE.fm Page 11 Friday, September 19, :49 PM Typical data (premixed reagents, see section 9). Description Concentration cpm % B/Bo Total activity (T) Zero calibrator (Bo) 0 pg/ml (0 pmol/l) Calibrator 1 10 pg/ml (37 pmol/l) Calibrator 2 40 pg/ml (147 pmol/l) Calibrator pg/ml (367 pmol/l) Calibrator pg/ml (1468 pmol/l) Calibrator pg/ml (3670 pmol/l) Tracer binding, % B/Bo Fig. 1 - Example of calibration curves: Routine assay 25 µl sample Premixed reagents Concentration of estradiol, pg/ml 11

12 EstradCE.fm Page 12 Friday, September 19, :49 PM 11. LIMITATIONS OF THE PROCEDURE General remarks Diagnosis should not be established on the basis of a single test result, but should be determined in conjunction with clinical findings and other diagnostic procedures as well as in association with medical judgement. Bacterial contamination or repeated freeze-thaw cycles of the specimens may affect the assay results. A skillful technique and strict adherence to the instructions are necessary to obtain reliable results. In particular, precise pipetting and accurate aspiration are essential. Non-reproducible results may arise from methodological factors, such as: cross-exchange of vial caps use of the same tip when withdrawing from different vials or dispensing different samples leaving the vials open for long exposure of reagents or samples to intense heat or heavy sources of bacterial contamination inadequate aspiration of incubation mixture contamination of tube rims by tracer or samples casual oscillations or inadequate handling of the gamma counter use of reagents from different master batches. Compatibility of different assay procedures Care should be taken not to mix the three assay procedures indicated before. Specifically, it is not possible to read a 25 µl sample off a 50 µl calibration curve. If a smaller sample must be used in the routine system the difference of volume has to be made up by the addition of zero calibrator (see section 8). Logit/log data reduction of assay results From most of the assay data it is evident, that in logit/log transformation the calibration curve is not along a straight line. Usually the central calibrators (e.g. 400 pg/ml, 1000 pg/ml) give lower tracer binding than expected. RIA calibration curves only follow a straight line if the assay reactions are in perfect accordance with a simplified theoretical model. In reality, deviations may occur. The complex nature of the reactions in these systems represents such a case. Deviations from straight lines occur and cannot be avoided. Some data processing facilities use logit/log transformations as the mathematical basis for computation of results. Although the merits of this type of program are fully acknowledged, more modern programs (four parameters, spline, multiple binding site program) give results that more fully appreciate real (rather than theoretical) RIA situations. The use of such programs is therefore advised. 12

13 EstradCE.fm Page 13 Friday, September 19, :49 PM 12. SPECIFIC PERFORMANCE CHARACTERISTICS Analytical specificity Analytical specificity may be defined as the ability of the assay to accurately detect specific analyte in the presence of potentially interfering factors in the sample matrix (e.g., anticoagulants, haemolysis, effects of sample treatment), or cross-reactive analytes. Interference. Controlled studies of potentially interfering substances or conditions showed that the assay performance was not affected by anticoagulants (citrate, EDTA, heparin), haemolysis (up to 200 mg/dl haemoglobin), lipaemia (up to 500 mg/dl triglycerides), bilirubinaemia (up to 20 mg/dl bilirubin) or one freeze-thaw cycle of samples. Cross-reactions. Data on the specificity of the antiserum used in this kit are expressed as the ratio of estradiol concentration to the cross-reacting hormone concentration at 50% inhibition of maximum binding as summarized below. Hormone Cross-reaction Estradiol 1.0 Estrone Estriol Ethinylestradiol < Progesterone < Testosterone < Androstenediol < Estradiol-3-glucuronide Estradiol-17-glucuronide Analytical sensitivity Analytical sensitivity may also be expressed as the limit of detection, which is the minimal amount of specific analyte detectable by the assay. The limit of detection is 2 pg/ml at 95% confidence limit (8). This was calculated as the apparent concentration of analyte which was distinguishable from the zero calibrator, that is, two standard deviations below zero. 13

14 EstradCE.fm Page 14 Friday, September 19, :49 PM Precision Different sample pools, containing different concentrations of specific analyte, were assayed to determine repeatability and reproducibility of the assay (i.e., within- and between-assay variability). Repeatability A B C Number of determinations Mean (pg/ml) Standard deviation Coefficient of variation (%) Reproducibility A B C Number of determinations Mean (pg/ml) Standard deviation Coefficient of variation (%) Trueness Recovery test. A serum with low estradiol concentration was tested as such and after mixing with increasing amounts of estradiol. Percent recovery is calculated as recovered/ expected value ratio x 100. Added concentration, pg/ml Expected concentration, pg/ml Measured concentration, pg/ml % Recovery

15 EstradCE.fm Page 15 Friday, September 19, :49 PM Dilution test. Two sera with high estradiol concentration were tested after serially diluting with the zero calibrator. Dilution Expected concentration, pg/ml Measured concentration, pg/ml % Recovery neat : : : : neat : : : : : EXPECTED VALUES The normal ranges found with this kit are summarized in the following table. The values have been determined on specimens from healthy individuals. Each laboratory is advised to establish its own normal ranges. Subjects No. of samples Concentration (pg/ml) Concentration (pmol/l) Prepubertal children Males Early follicular phase Preovulatory peak Luteal phase Post menopausal 49 < 25 < 92 Pregnant women: 222 1st trimester nd trimester rd trimester

16 EstradCE.fm Page 16 Friday, September 19, :49 PM A representative profile of estradiol concentrations found in a normal menstrual cycle is displayed in Fig Concentration of estradiol in serum, pg/ml Days of cycle Fig. 2 - Concentration of estradiol in serum measured with this method during a representative menstrual cycle. 16

17 EstradCE.fm Page 17 Friday, September 19, :49 PM Definition of units of radioactivity (Ci, Bq) 1 Curie = 1 Ci = 3.7 x disintegrations/second 1 Béquerel = 1 Bq = 1 disintegration/second 1 µci = Bq = 37 kbq. Calculation of centrifugal force Please use the nomogram to find rpm s to corresponding g -values (see Fig. 3). Measure the radius in cm from the axis to the middle of the spinning tubes. Trace a line from the measured radius through the desired g -value. You will find the necessary rpm s on the corresponding scale. Example: Radius (axis of rotor to center of tube) = 15 cm Required g -force = 2000 g Result = approx rpm. rpm g Radius, cm Fig. 3 - Nomogram for the calculation of centrifugal force

18 EstradCE.fm Page 18 Friday, September 19, :49 PM 14. WARNINGS AND PRECAUTIONS Test components contain sodium azide as a preservative. Because sodium azide may form explosive lead or copper azide in plumbing, it is recommended that drains be thoroughly flushed with water after disposal of solutions containing sodium azide (Council Directive 99/45/EC). R 20/21/22 Harmful by inhalation, in contact with skin and if swallowed. R 32 Contact with acids liberates very toxic gas. S 28 After contact with skin, wash immediately with plenty of water. Sodium merthiolate (Council Directive 99/45/EC): R 20/21/22 Harmful by inhalation, in contact with skin and if swallowed. R 33 Danger of cumulative effects. S 28 After contact with skin, wash immediately with plenty of water. S 36 Wear suitable protective clothing. S 45 In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible). All serum and plasma units used to produce the components provided in this kit have been tested for the presence of HBsAg, anti-hcv, and anti-hiv-1/2 and found to be nonreactive. As, however, no test method can offer absolute assurance that pathogens are absent, all specimens of human origin should be considered potentially infectious and handled with care. 15. SAFETY PRECAUTIONS Do not eat, drink, smoke or apply cosmetics in the assay laboratory. Do not pipette solutions by mouth. Avoid direct contact with all potentially infectious materials by using protective clothing such as lab coats, protective glasses and disposable gloves. Wash hands thoroughly at the end of each assay. Avoid splashing or forming an aerosol. Any reagent spills should be washed with a 5% sodium hypochlorite solution and disposed of as though potentially infectious. All samples, biological reagents and materials used in the assay must be considered potentially able to transmit infectious agents. They should therefore be disposed of in accordance with the prevailing regulations and guidelines of the agencies holding jurisdiction over the laboratory, and the regulations of each Country. Disposable materials must be incinerated; liquid waste must be decontaminated with sodium hypochlorite at a final concentration of 5% for at least half an hour. Any materials to be reused must be autoclaved using an overkill approach (USP 24, 2000, p. 2143). A minimum of one hour at 121 C is usually considered adequate, though the users must check the effectiveness of their decontamination cycle by initially validating it and routinely using biological indicators. 18

19 EstradCE.fm Page 19 Friday, September 19, :49 PM 16. BASIC RULES OF RADIATION SAFETY This 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. Its receipt, acquisition, possession, use and transfer are subject to the regulations of each Country. Adherence to the basic rules of radiation safety should provide adequate protection. Do not eat, drink, smoke or apply cosmetics where radioactive materials are used. Do not pipette radioactive solutions by mouth. Avoid direct contact with all radioactive materials by using protective articles such as lab coats and disposable gloves. All radiological work should be done in a designated area away from traffic. Radioactive materials should be stored in their original containers in a designated area. A record book for logging receipt and disposal of all radioactive materials should be kept. Laboratory equipment and glassware which are subject to contamination should be segregated to prevent cross-contamination of different radioisotopes. Any radioactive spills should be taken care of immediately in accordance with established procedures. All radioactive materials must be disposed of in accordance with the prevailing regulations and guidelines of the agencies holding jurisdiction over the laboratory. 19

20 EstradCE.fm Page 20 Friday, September 19, :49 PM KIT PER IL DOSAGGIO RADIOIMMUNOLOGICO DIRETTO DEL 17 BETA-ESTRADIOLO IN CAMPIONI DI SIERO O PLASMA UMANO 1. INTRODUZIONE Il 17 beta-estradiolo è un ormone steroideo femminile secreto dalle ovaie di peso molecolare circa 272 dalton. La funzione principale dell estradiolo consiste nel predisporre la mucosa uterina allo stadio pregestativo. Tale ormone inoltre inibisce la produzione di ormone follicolo-stimolante (FSH) e stimola la liberazione preovulatoria di ormone luteinizzante (LH) da parte dell ipofisi (1). Nel siero l estradiolo è complessato a una proteina vettrice (SHBG = sex hormone binding globulin) (5, 6). Questa proteina, come nel caso di altri ormoni a basso peso molecolare, può intervenire nella regolazione della liberazione dell ormone a livello degli organi bersaglio (7). Nelle donne non gravide i livelli sierici di estradiolo presentano un andamento ciclico con un massimo che si verifica poco prima dell ovulazione (2). La determinazione delle concentrazioni di estradiolo è utile per valutare disfunzioni della mestruazione, sindrome femminilizzante negli adolescenti e tumori secernenti estrogeni. L estradiolo può inoltre risultare elevato nella ginecomastia e nella cirrosi. Viene spesso determinato nel corso di terapie per la sterilità, quali stimolazione dell ovulazione mediante clomifene o tecniche di fecondazione in vitro (FIV) (3). I livelli di estradiolo sierico raggiungono concentrazioni molto elevate durante la gravidanza. Pertanto il controllo dei livelli di estradiolo fornisce utili indicazioni sulla crescita fetale (4). Nei maschi normali i livelli di estradiolo sono molto bassi. Livelli anormalmente elevati possono essere indicativi di tumori testicolari (1). 2. FINALITÀ D USO I reattivi forniti nel kit sono solo per uso diagnostico in vitro. Questo kit deve essere impiegato per la determinazione quantitativa diretta dei livelli di 17 beta-estradiolo nel siero e nel plasma umani. L intervallo di dosaggio e le prestazioni del metodo sono stati ottimizzati per consentire la determinazione di livelli di estradiolo sia molto bassi (p.es. negli adolescenti) sia molto elevati (p.es. nella FIV). 3. PRINCIPIO DEL DOSAGGIO Il metodo per la determinazione diretta dell estradiolo si basa sul principio del dosaggio radioimmunologico competitivo. I campioni e i calibratori vengono incubati con estradiolo marcato con 125 I e con un anticorpo anti-estradiolo. Dopo una seconda incubazione seguita da una centrifugazione, si ottiene la separazione del tracciante non legato all anticorpo 20

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