Explaining Recent Declines in Adolescent Pregnancy in the United States: The Contribution of Abstinence and Improved Contraceptive Use| John S. Santelli, MD, MPH, Laura Duberstein Lindberg, PhD, Lawrence B. Finer, PhD, and Susheela Singh, PhDIn recent years, the United States has had the Objectives. We explored the relative contributions of declining sexual activityhighest rate of adolescent
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IntroductionPATHOZYME PROGESTERONE Ref OD487
Enzyme Immunoassay for the quantitative determination of Progesterone
in human serum or plasma.
Store at 2oC to 8oC. DO NOT FREEZE.
For in-vitro use only.
MATERIAL REQUIRED BUT NOT PROVIDED
Progesterone is a C21 steroid which is synthesised from both Micropipettes: 100l, 200l, 1000l and 5000l tissue and circulating cholesterol. Cholesterol is transformed to pregnenolone which is then converted via a combined dehydrogenase and isomerase to progesterone. The principle Microplate reader fitted with a 450nm filter production sites are the adrenals and ovaries and the placenta during pregnancy. The majority of this steroid is metabolised in the liver to pregnanediol and conjugated as a glucuronide prior PRECAUTIONS
Progesterone exhibits a wide variety of end organ effects. The primary role of progesterone is exhibited by the reproductive PATHOZYME PROGESTERONE contains materials of human
organs. In males, progesterone is a necessary intermediate for origin which have been tested and confirmed negative for HCV, the production of cortcosteroids and androgens. In females, HIV I and II antibodies and HBsAg by FDA approved methods progesterone remains relatively constant throughout the at single donor level. Because no test can offer complete follicular phase of the menstrual cycle. The concentration then assurance that products derived from human source will not increases rapidly following ovulation and remains elevated for transmit infectious agents it is recommended that the reagents 4-6 days and decreases to the initial level 24 hours before the within this kit be handled with due care and attention during onset of menstruation. In pregnancy, placental progesterone use and disposal. All reagents should, however, be treated as production rises steadily to levels of 10 to 20 times those of the potential Biohazards in use and for disposal. Do not ingest. Progesterone measurements are thus performed to determine PATHOZYME PROGESTERONE Reagents do not contain
ovulation as well as to characterise luteal phase defects. dangerous substances as defined by current UK Chemicals Monitoring of progesterone therapy and early stage pregnancy (Hazardous Information and Packaging for Supply) regulations. evaluations comprise the remaining uses of progesterone All reagents should, however, be treated as potential biohazards in use and disposal. Final disposal must be in INTENDED USE
PATHOZYME PROGESTERONE is an Enzyme Immunoassay
PATHOZYME PROGESTERONE Stop Solution is dilute
(EIA) for the quantitative determination of total Progesterone in Hydrochloric Acid and is therefore corrosive. Handle with care. In case of contact, rinse thoroughly with water. PATHOZYME PROGESTERONE reagents contain 1.0%
PRINCIPLE OF THE TEST
Proclin 300* as a preservative which may be toxic if ingested. In case of contact, rinse thoroughly with water and seek The PATHOZYME PROGESTERONE is based on the
principle of competitive binding between Progesterone in the test specimen and Progesterone-HRP Conjugate for a constant amount of rabbit anti-Progesterone. In the incubation, goat * Proclin 300 is a Trade Mark of ROHM and HAAS Ltd. anti-rabbit IgG-coated wells are incubated with Progesterone standards, controls, patient samples, Progesterone-HRP Conjugate Reagent and rabbit anti-Progesterone Reagent. Reagents must be stored at temperatures between 2oC to 8oC. During the incubation, a fixed amount of HRP-labelled Progesterone competes with the endogenous Progesterone in Expiry date is the last day of the month on the bottle and the kit the standard and sample or quality control serum for a fixed number of binding sites of the specific Progesterone antibody. label. The kit will perform within specification until the stated Thus, the amount of Progesterone peroxidase conjugate expiry date as determined from date of product manufacture immunologically bound to the well progressively decreases as and stated on kit and components. Do not use reagents after the concentration of Progesterone in the specimen increases. Unbound Progesterone peroxidase conjugate is then removed and the wells washed. The Substrate (TMB) is then added, Exposure of reagents to excessive temperatures should be resulting in the development of blue colour. The colour avoided. Do not expose to direct sunlight. development is stopped with the addition of stop solution, and DO NOT FREEZE ANY OF THE REAGENTS as this will cause the absorbance is measured spectrophotometrically at 450nm. The intensity of the colour formed is proportional to the amount of enzyme present and is inversely related to the amount of SPECIMEN COLLECTION AND PREPARATION
unlabelled Progesterone in the sample. A standard curve is obtained by plotting the concentration of the standard versus the absorbance. The Progesterone concentration of the specimens and controls run concurrently with the standards Obtain a sample of venous blood from the patient and allow a can be calculated from the standard curve. clot to form and retract. Centrifuge clotted blood sample and This test has been calibrated against in house standards. collect clear serum. Fresh serum samples are required. There is no International standard for this test. Obtain a sample of venous blood from the patient and add to EDTA blood collection vial. Centrifuge sample and collect clear plasma. Fresh plasma samples are required. Do not use haemolysed, contaminated or lipaemic serum or plasma for testing as this will adversly affect the results. Microtitre Plate
12 x 8 wells x 1
Serum or plasma may be stored at 2oC to 8oC for up to 48 Breakable wells coated with Goat anti Rabbit IgG contained in a hours prior to testing. If longer storage is required, store at – 20oC for up to 1 year. Thawed samples must be mixed prior to Reference Standard: Human serum free of Progesterone. Do not use Sodium Azide as a preservative as this may inhibit 0.5 ng/ml
Reference Standard: Progesterone diluted in human serum. Do not repeatedly freeze-thaw the specimens as this will cause 3.0 ng/ml
Reference Standard: Progesterone diluted in human serum. REAGENT PREPARATION
All reagents should be brought to room temperature (20oC to Reference Standard: Progesterone diluted in human serum. 25oC) and mixed gently prior to use. Do not induce foaming. Working Solution: Dilute the concentrated conjugate using 1 part conjugate to 10 parts conjugate diluent ( 1/11 dilution ) Reference Standard: Progesterone diluted in human serum. Ready to use (Colourless) 100l is required per well. Diluted reagent is stable at 2oC to Reference Standard: Progesterone diluted in human serum. LIMITATIONS OF USE
Level as stated on vial
The use of samples other than serum and EDTA plasma have not Known level of Progesterone diluted in human serum. been validated in this test. There is no reuse protocol for this product. When making an interpretation of the test it is strongly advised to take all clinical data into consideration. Diagnosis should Level as stated on vial
not be made solely on the findings of one clinical assay. Known level of Progesterone diluted in human serum. Ready to use (Colourless) Progesterone
Rabbit anti Progesterone reagent. Ready to use. (Pink)
Progesterone conjugated to horseradish Peroxide
Ready to use. (Blue)
Phosphate based buffer containing stabilising proteins. Ready to use. ( Blue ) Substrate Solution: 3,3’, 5,5’ Tetramethyl Benzidine in a citrate buffer. Ready to use. (Colourless) Stop Solution: Hydrochloric Acid diluted in purified water.
Ready to use. (Colourless)
Instruction leaflet and EIA Data Recording Sheet 1 + 1
Bring all the kit components and the test sample to room temperature (20C to 25C) prior to The lowest detectable level of Progesterone in this test is approximately 0.0625ng/ml One set of Standards should be run with each batch of test sample. Secure the desired SPECIFICITY
number of coated wells in the holder. Record the position of the standards and the test samples on the EIA Data Recording Sheet provided. The following materials have been checked for cross reactivity. The percentage indicates cross Unused strips should be resealed in the foil bag containing the desiccant, using the resealing reactivity at 50% displacement compared to Progesterone. zip-lock before being replaced at 2C to 8C. Data on the cross-reactivity for several endogenous and pharmaceutical steroids are summarised in Dispense 25l of standards, test samples and controls into the appropriate wells. Dispense 100l working solution of Progesterone-HRP Conjugate Reagent to each well. Cross-reactivity(%) = Observed Progesterone Concentrationx100 Dispense 50l of rabbit anti-Progesterone Reagent to each well. Thoroughly mix for 30 seconds. It is very important to mix completely. Incubate at room temperature (20oC to 25 oC) for 90 minutes. Cross-reactivity
At the end of the incubation period, discard the contents of the wells by flicking plate contents into a Biohazard container. Then strike the wells sharply against absorbent paper. Ensure adequate disinfectant is contained in the Biohazard container. Hand Washing: Fill the wells with a minimum of 300l of distilled water per well. Flick plate contents into a Biohazard container. Then strike the wells sharply against absorbent paper. Strike the wells sharply onto absorbent paper or paper towel to remove all residual water Machine Washing: Ensure that 300l of distilled water is dispensed per well and that an appropriate disinfectant is added to the waste collection bottle. Wash the empty wells 5 times. After washing remove excess fluid by striking the wells sharply onto absorbent paper or paper EVALUATION DATA
towel to remove all residual water droplets. Dispense 100l of Substrate solution into each well. Gently mix for 5 seconds. Calibrated to major competitors and in house standards. Incubate in the dark at room temperature (20C to 25C) for 20 minutes. The co-efficient of variation of PATHOZYME PROGESTERONE is less than or equal to 10%
Stop the reaction by adding 100l of Stop Solution to each well. Gently mix for 30 seconds. It is important to make sure that all the blue colour changes to In an evaluation between the Omega Pathozyme Progesterone kit and the DRG BIOC Kit for samples with levels between 0.35 ng/ml and 73.83 ng/ml the following data was generated. Read the absorbance at 450nm with a microtitre well reader within 10 minutes. TROUBLESHOOTING
For use by operatives with at least a minimum of basic laboratory training. Do not use damaged or contaminated kit components. Use a separate disposable tip for each sample to prevent cross contamination. Duplication of all standards and specimens, although not required, is recommended. These kits were shown to give good correlation. Specimens and standards should be run at the same time to keep testing conditions the same. REFERENCES
It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used, Radwanska, E., Frankenberg, J., and Allen. E., Plasma Progesterone Levels in Normal and
since pipetting of all Standards and specimens should be completed within 3 minutes. A full plate of 96 Abnormal Early Pregnancy, Fertility and Sterility 30, 398-402 (1978). wells may be used if automated pipetting is available. Autrere, M.B., and Benson, H., Progesterone: An Overview and Recent Advances, J. Par.
Replace caps on all reagents immediately after use. March, C.M., Goebelsmann, U., Nakamura, R.M., and Mischell, D.R. Roles of Estradiol and
Avoid repeated pipetting from stock reagents as this is likely to cause contamination. Progesterone in Eliciting the Midcycle Luteinising Hormone and Follicle-Stimulating Hormone Surges. J. Clin. Endo. And Metab. 48: 507-513, (1979). Do not mix reagents or antibody coated strips from different kits. When dispensing, care should be Ross, G.T., Van De Wiele, R.L., and Frantz, A.G. The Ovaries and the Breasts in Textbook
taken not to touch the surface of the well. of Endocrinology, R.H. Williams ed. P355-407, W.B. Saunders, Phil. (1981). Chattoraj, S.C., Endocrine Function in Fundamentals of Clinical Chemistry, N.W. Tietz ed.
Do not allow reagent to run down the sides of the well. Prior to the start of the assay bring all reagents p699-823, W.B. Saunders, Phil. Chap. 13 (1976). to room temperature (20oC to 25oC). Gently mix all reagents by gentle inversion or swirling. Shepard, M.K., and Fainstat, T. Comparison of Serum Progesterone and Endometrial Biopsy
for Confirmation of Ovulation and Evaluation of Luteal Function. Fertility and Sterility, 28: 541; Once an assay has been initiated, the wells should not be allowed to become dry during the assay. Johansson, E.D., and Johansson, L.E. Progesterone Levels in Amniotic Fluid and Plasma
Do not contaminate the Substrate Solution as this will render the whole kit inoperative. from Women. I. Levels During Normal Pregnancy. Acta. Obstet. Gynaecol. Scand. 50: 339; Check the precision and accuracy of the laboratory equipment used during the procedure to ensure USA Centre for Disease Control/National Institute of Health Manual “Biosafety in Microbiological and Biomedical Laboratories” 1984. The unused strips should be resealed in the foil bag, containing the desiccant, using the resealing zip- lock before being replaced at 2oC to 8oC. QUICK REFERENCE TEST PROCEDURE
CALCULATION OF RESULTS
Dispense 25l of standards, test samples, controls. Calculate the mean absorbance value (A450) for each set of Standards, Controls and samples. Add 100l working solution Progesterone HRP conjugate to each well. Construct a point to point standard curve by plotting the mean absorbance obtained for each Standard against its concentration in ng/ml on graph paper, with absorbance values on the vertical or Y-axis and Add 50l of Rabbit anti-Progesterone into each well. Gently mix for 30 seconds. concentrations horizontal or the X-axis. Use the mean absorbance values for each specimen to determine the corresponding concentration of Incubate for 90 minutes at room temperature (20oC to 25oC) Progesterone in ng/ml from the standard curve. If levels of controls or users known samples do not give expected results, test results must be Discard the well contents and wash 5 times with distilled water. If using a software package choose a polygon with data extrapolation curve fit. Add 100l of substrate solution into each well and gently mix for 5 seconds. EXPECTED VALUES AND SENSITIVITY
Incubate in the dark for 20 minutes at room temperature (20oC to 25oC). The graph produced by the calibrators should be Hyperbolic in shape with the OD450 of the calibrators Add 100l Stop Solution to each well and gently mix for 30 seconds. inversely proportional to their concentration. The OD of calibrator A should be greater than 1.5 and the OD of calibrator F less than 0.75 for the assay results to be valid. Read the Optical Densities immediately (no later than 10 minutes) using a microplate Each laboratory should establish its own normal range based on the patient population. PATHOZYME PROGESTERONE was performed on randomly selected outpatient clinical laboratory
The results of these determinations are as follows: OMEGA DIAGNOSTICS LTD.
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tected by echocardiography after 20 weeks’ gestation14. Meyers DG, Meyers RE, Prendergast TW. The usefulness of diagnosticand is normally 2 mm or less in depth. More ﬂuid shouldtests on pericardial ﬂuid. Chest 1997;111(5):1213–21. 15. Eisenberg MJ, Dunn MM, Kanth N et al. Diagnostic value of chestraise questions of hydrops foetalis, Rh disease, hypoal-buminemia, and immunopathy or ma