Human FSH (Menopause) (midstream) Rapid Test (RAP-4155) For the rapid detection of Follicle Stimulating Hormone (hfsh) in human urine specimens

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1 Human FSH (Menopause) (midstream) Rapid est (RAP-4155) For the rapid detection of Follicle Stimulating Hormone (hfsh) in human urine specimens For in vitro diagnostic use. INENDED USE he DRG FSH Rapid est is intended for a visual, qualitative determination of human Follicle Stimulating Hormone (FSH) in human urine at a sensitivity of 25 miu/ml for the confirmation of hormone changes related to symptoms associated with the stages of menopause. INRODUION Follicle Stimulating Hormone is a peptide hormone produced in the pituitary gland of the brain. It is normally present in the blood or urine varying in concentration with the stage of the menstrual cycle. When estrogen levels drop, FSH is released from the pituitary gland indicating that either a woman in mid-menstrual cycle or indicating the onset of perimenopause. During early menopause, changes take place in the balance of hormones that regulate and control menstrual cycles. As a woman grows older and passes out of childbearing stage of life, the ovaries gradually make less of the hormone estrogen and FSH increases. FSH normally regulates the growth and development of an egg. Once this part of the monthly cycle is complete, FSH production is stopped and it returns to normal. As the body decreases estrogen production with age, more FSH is made. Over time these hormone changes cause menstrual periods to stop completely and "menopause" has occurred. he slow change in ovary function can happen between 2 and 10 years before the final period. his early stage before menopause is called Perimenopause. During this stage, the levels of FSH may rise to positive levels and slowly return to normal, causing irregular or missed periods. he testing for FSH should, therefore, be performed twice to help identify the levels of FSH throughout a menstrual cycle. he one-step FSH est is a chromatographic immunoassay (IA) for the rapid qualitative determination of FSH in the urine. he immunological specificity of the test kit virtually eliminates cross reactivity and interference to structurally related glycoprotein hormones such as hlh, hg and hsh. PRINIPLE he DRG FSH Rapid est is a qualitative, two site sandwich immunoassay for the determination of human follicle stimulating hormone (FSH) in urine specimens. he membrane was precoated with FSH specific antibodies on the test region. During the test, the specimen is allowed to react with the FSH monoclonal antibody-colloid gold conjugate which was pre-dried on the test strip. he mixture then moves upward on the membrane chromatographically by the capillary action. For a positive specimen, the conjugate binds to the FSH forming an antibody-antigen complex. his complex binds to the FSH antibody as a capture regents on the test region and produces a colored band when FSH concentration is equal to or greater than 25 miu/ml. he DRG FSH Rapid est also provides a built-in process control. A colored band should always appear in the ontrol Region, regardless of the presence of any urinary FSH. his control band verifies that 1) sufficient urine volume was added, 2) proper urine flow was obtained and 3) that the test is functioning correctly. he absence of a control band may indicate the test was not performed correctly or that the test did not function correctly. MAERIALS SUPPLIED 1

2 Human FSH (Menopause) (midstream) Rapid est (RAP-4155) 1. est device containing sample reaction unit, pnk-colored colloidal gold conjugate unit, ad a chromatographic membrane midstream wand unit. 2. est instructions for the DRG FSH Rapid Midstream est midstream device. MAERIALS REQUIRED 1. lean urine sample container 2. imer or clock with setting of ten minutes SORAGE AND SABILIY he DRG FSH Rapid est can be stored refrigerated or at room temperature (2-28 º) in sealed pouch. Avoid freezing. PREAUIONS For in vitro diagnostic use For professional use only Read instructions carefully before performing test If the laboratory modifies the test system instructions, the test is considered High omplexity and subject to all applicable LIA requirements. Do not use after expiration date. est device should remain sealed until ready for use. Urine specimens may be potentially infectious. Avoid contact with skin by wearing gloves and proper laboratory attire. Properly handle and dispose of all used test devices in an appropriate biohazard container. he reagents in this kit contain sodium azide which may react with lead or copper plumbing to form potentially explosive metal azide build up. Specimens should be considered hazardous and handled appropriately. If specimens are to be shipped, they should be packed in compliance with regulations covering the transportation of etiologic agents. SPEIMEN OLLEION AND HANDLING ollect and store specimens following standard clinical procedures: he urine specimen must be collected in a clean, dry container either plastic or glass, without preservatives. No centrifugation or filtration of urine is required. However, if the performance of the test is hindered by high turbidity or presence of particulates in the specimen, it should be centrifuged to remove solids prior to performing the test. he test can be performed at anytime during the day, however, for best results, the urine sample for the test should be collected at about the same time each day. It is highly recommended that the first morning urine be tested, since it generally contains the highest concentration of FSH. Urine collected during the day may dilute the FSH level and may cause a false negative result. If specimens cannot be tested after collection, they should be stored refrigerated at 2-8 º for 24 hours. If samples are refrigerated, they must be equilibrated to room temperature before testing. If testing is delayed more than 24 hours the specimen should be frozen at 20 º. Do not use a frost-free freezer, 2

3 Human FSH (Menopause) (midstream) Rapid est (RAP-4155) which may allow the specimens to go through freeze-thaw cycles that may denature the FSH and cause spurious results. he frozen specimen must be thawed, brought to room temperature and thoroughly stirred before testing. Avoid repeated thawing and freezing. Urine specimens may be potentially infectious. Wear gloves and proper laboratory attire to avoid contact with skin. Dispose of all test devices in approved biohazard containers. Residual urine should be disposed of in a medically approved manner after completion of testing. ASSAY PROEDURE Remove the test from protective pouch by tearing along the notch. Do not open foil pouch until ready to begin testing. Samples must be equilibrated to room temperature (15-28 ) before testing. Remove the cap and place the exposed absorbent wick directly into the urine stream or directly into a collection cup containing the urine specimen for 10 seconds or until the absorbent wick is saturated with urine. Remove the test from the urine stream, replace the cap and place on a flat, clean surface. If the flow of the specimen is not observed through the test area, this may be due to an insufficient amount of specimen absorbed on the wick. he person performing the test should review the instructions thoroughly prior to performing the test again. he test should be repeated using a new test device. Read the test result at 5 minutes. Do not read the test after ten minutes. Note: o avoid confusion, discard the test device after interpreting results. Each test should be used only once. INERPREAION OF RESULS Negative: Only one colored band appears on the control region (). No colored band in the test region (). Positive: In addition to the control band, a distinct colored band also appears in the test region (). Invalid: If no bands appear after 10 minutes, or if no band appears in the control region () the result is invalid. he protocol may not have been performed correctly, or the test is deteroirated. he assay should be repeated using a new test device. Note: he test band intensity may be stronger or weaker than that of the control band. A very faint band in the test region indicates that the concentration of FSH is near the detection level in the urine. he sample should be re-tested or confirmed with a more specific method before a positive determination is made. Positive ( + ) Negative ( - ) Invalid 3

4 Human FSH (Menopause) (midstream) Rapid est (RAP-4155) 1S est 2nd est INERPREAION Irregular ycles plus menopausal symptoms Positive May be perimenopausal but should repeat test and consult physician Positive Positive May be experiencing perimenoupause; consult physican Positive Negative May be experiencing perimenopause; consult physician Negative Appears to not be perimenopausal this cycle Negative Negative Although results are negative, symptoms suggest perimenopause may still be considered. Other causes of irregular cycles should be sought. Discuss results and symptoms with physician. Negative Positive May be experiencing perimenopause; consult physician Experiencing symptoms, but NO menstrual period for 12 months Positive Appears that menopause has occurred, repeat test to confirm result. onsult physician. Negative Although result is negative, symptoms suggest that menopause may have occurred. onsult physician for futher testing. QUALIY ONROL Good laboratory practice includes the use of control to ensure proper test performance and reliability. he DRG FSH Rapid Midstream est has been standardized to World Healht Organization First International Reference Preparation. Quality control standards are available for the valication of device functionality from commercial sources such as Sigma or Bio-Rad. FSH negative and positive controls are available from commercial sources. When testing quality control standards, use the same assay procedure as with a urine sample. It is recommended that control specimens be used for each new lot of kits. Each laboratory should establish their own criteria for interpretation of results as baseline FSH levels and patterns of FSH secretion can vary among individuals. his should be based on the following parameters: 1) each new lot or shipment of devices received, 2) each newly opened kit (2 tests), 3) each new operator (an individual that has not run the tests for two weeks), 4) weekly, as a continued check on storage conditions, 5) whenever problems are identified (e.g., storage, operator error, or other), or 6) other frequency as required by individual laboratory Quality ontrol procedures. he use of control samples is advised to assure the performance of the test and reactivity of the reagents. It is recommended that the laboratory prepare its own urine pools using known menopausal urine (30-50 miu/ml) as a positive, and a pre-menopause or pregnant female urine (<15 miu/ml) as a negative. Alternatively, the use of serum or urine commercial controls with assayed values, e.g. DRG Immunoassay Plus ontrol (Bio-Rad), should be run to ensure adequate performance of the test. PERFORMANE HARAERISIS he DRG FSH Urine Rapid Menopause est is capable of confirming elevated levels of FSH caused by the stages of menopause. Naturally occurring perimenopause typically falls between 40 and 55 years of age. In studies performed with the DRG FHS Urine Rapid Menopause est on women between the ages of 13 and 80, positive FSH levels were identified as early as years of age. Negative results were found for women of years of age. Negative results may generally be obtained at the age of 50 or above. 4

5 Human FSH (Menopause) (midstream) Rapid est (RAP-4155) EXPEED WAIVER PERFORMANE able 1, below, describes the expected waiver performance for clinical tests performed on 247 participants: 247 laypersons and 2 technicians. able 1: Expected Waiver Performance: Layperson and echnician Results DRG Menopause est (+) ( - ) Row otals Laypersons echnicians Note: One sample was identified as positive by the layperson and negative by the technician. he expected waiver positive results were 117/118 or 99.2% accuracy. he expected waiver negative results were 129/130 or 99.2%. he overall relative expected accuracy was 246/247 or 99.6%. OMPARAIVE REFERENE SUDY he DRG FSH Urine Rapid Midstream est was compared to a reference EIA test for human serum samples. A total of 247 men and women were tested by the two methods. he DRG test was interpreted by both the layperson and the technician. At two testing sites, results interpretation for one test was different between the technician and the layperson. In addition to outside studies, 169 urine samples were obtained and tested in-house and compared to the reference EIA test. able 2 summarizes results from the 416 total samples obtained for comparison to the reference EIA test. able 2: ombined Results: DRG Menopause est vs. Serum EIA DRG Menopause est Serum EIA (+) ( - ) Row otals (+) ( - ) olumn otals When compared to Serum EIA est data, the relative sensitivity or percent agreement with the DRG FSH Urine Rapid Midstream est positive samples with the external clinical study was 182/184 or 98.9%. Negative samples recovered a relative specificity of agreement of 231/233 or 99.1%. he overall relative accuracy obtained was 413/416 or 99.0%. SENSIIVIY he DRG FSH Urine Rapid Midstream est is designed for the detection of hfsh in human urine at the cut-off detection level of 25mIU/ml. o verify test sensitivity, a standard panel of FSH was prepared by spiking fresh normal human pooled urine specimens with FSH standard at concentrations of 0, 5, 10, 15, 20, 25, 50 and 100 miu/ml. his procedure tested 50 devices per standard level. All samples at concentrations equal to or below 20mIU/ml tested negative. All samples at concentrations equal to or above 25mIU/ml tested positive. he sensitivity of the DRG FSH Urine Rapid Midstream est was thus determined to be 25mIU/ml. Results per FSH standard concentration are tabulated on able 3, below. 5

6 Human FSH (Menopause) (midstream) Rapid est (RAP-4155) able 3: Sensitivity Study results for DRG FSH midstream device DRG Menopause FSH oncentration(miu/ml) est Qty. ested Positive Negative SPEIFIIY ross-reactivity esting: he specificity of the DRG FSH Urine Rapid Midstream est was determined from cross reaction studies with known amounts of human hormones commonly found in normal human urine. Human Luteinizing Hormone (hlh) 500 miu/ml Human horionic Gonadotropin Hormone (hg) 250 miu/ml Human hyroid Stimulating Hormone (hsh) 1000 miu/ml Interference esting: he following substances were shown not to interfere with the DRG FSH Urine Rapid Midstream est at or below the following concentrations. Acetaminophen Acetysalicylic Acid Ampicillin Ascorbic Acid Atropine affeine Gentesic Acid Glucose 2000 mg/dl Hemoglobin 1 mg/dl Human Serum Protein 2000 mg/dl etracycline Uric Acid 10 mg/dl REPRODUIBILIY WIHIN-LO REPRODUIBILIY: hree batches of negative, normal human urine were spiked with FSH standard at concentrations of 0, 10, 25, 50 and 100 miu/ml. hree groups of five devices from one lot were tested at each concentration for a total of 15 devices / concentration. Results clearly showed excellent repeatability for all positive and negative urine samples using one lot of DRG FSH Urine Rapid Midstream est devices. INER-LO REPRODUIBILIY: Negative, normal human urine was spiked with FSH standard at concentrations of 0, 10, 25, 50 and 100 miu/ml. he samples were blinded by dispensing the standard solutions into 60 letter-coded, labeled vials per concentration. 20 devices per concentration were tested for each lot. All 60 vials were used for testing over a period of four weeks. he results of these tests clearly demonstrate that 6

7 Human FSH (Menopause) (midstream) Rapid est (RAP-4155) there is no appreciable inter-lot variation when testing both positive and negative spiked samples across three different lots of DRG FSH Urine Rapid Midstream est devices. LIMIAIONS If a specimen is too dilute (i.e. low specific gravity), it may not contain representative levels of hfsh. he first morning urine should be obtained hours later and retested. he first morning urine be tested, since it generally contains the highest concentration of FSH. hfsh concentrations less than 25 miu/ml will be detected as negative. Oral contraceptives, hormone replacement therapy, and estrogen supplements may affect FSH levels and could yield a false negative result. Ovarian and pituitary tumors can result in decreased FSH levels, which may cause a false negative result in the test. As is true with any diagnostic procedure, the physician should evaluate the data obtained by the use of this test in light of other clinical information. his test must not be used to determine fertility. ontraception decisions should not be made based on the results of this test. his test will not determine ovulation or pregnancy status. REFERENES AAE Medical Guidelines for linical Practice for Management of Menopause, Endocrine Practice, Vol. 5 No. 6, Nov / Dec Greendale, G., Lee, N., Arriola, E., he Menopause, he Lancet, Vol. 353, Fe b. 13, Mayeaux, E.J., Jr., Menopause/Perimenopause: Issues/Symptoms/ reatment, Lecture at Primary are in Women s Health Backer, l., et. al, Se rum Follicle Stimulating Hormone and Luteinizing Hormone Levels in Women aged in the U.S. Population: he hird National Health and Nutrition Examination Survey (NHANES III, ), Menopause, Vol. 6, No. 1,