I.

INTENDED USE

The timely and accurate diagnosis of rupture of fetal membranes (ROM) is crucial since the ROM may be associated with serious neonatal and maternal consequences. Failure to identify patients with ROM can result in the failure to intervene appropriately. Conversely, the false diagnosis of ROM can lead to inappropriate interventions (e.g., hospitalization or induction of labor). Therefore the correct and timely diagnosis of ROM is of crucial importance for the clinician. Accurate diagnosis of fetal membranes rupture, however, remains a frequent clinical problem in obstetrics.

The AmniSure test kit is a self-contained test system providing qualitative results that are both accurate and do not require collection methods such as speculum examination.

PRINCIPLE OF MEASUREMENT: 

The test does not require speculum examination that is used routinely today for ROM diagnosis. A sample of amniotic fluid (taken by vaginal swab) is placed into a vial with a solvent. The solvent extracts the sample from the swab for one minute, after which the swab is disposed. AmniSure Test strip, a lateral flow device, is then dipped into the vial. The sample substance flows from the Pad Region of the strip to the Test Region. The test result is indicated visually over the next 5-10 minutes by the presence of one or two lines. One line indicates no membranes ruptured, two lines indicates there is a rupture. The AmniSure® ROM Test uses the principles of immunochromatography to detect human PAMG-1 (placental α1-microglobulin) protein present in amniotic fluid of pregnant women. Placental Microglobulin was selected as a marker of fetal membranes rupture due to its unique characteristics, i.e. its high level in the amniotic fluid, low level in blood and extremely low background level (50-220 picogram/ml) in cervicovaginal secretions when the fetal membranes are intact.

The test employs highly sensitive monoclonal antibodies that detect even a minimum amount of the protein, which is present in cervico-vaginal secretions after the rupture of the fetal membranes. To minimize the frequency of false results, two monoclonal antibodies have been selected to set the sensitivity threshold of AmniSure® at the optimal low level. This level allows the detection of the extremely small quantities of amniotic fluid in vaginal secretions. Background concentration of PAMG-1 that uses this combination of monoclonal antibodies is around 50-220 picogram (i.e. 0.05-0.22 ng) per 1ml of vaginal secretion. The sensitivity cut-off of AmniSure® is 5 ng/ml, i.e. at least 20 times higher than the background concentration. This gap allowed increasing the accuracy of AmniSure®.

During the test procedure, placental microglobulin from the sample sequentially binds to monoclonal antibody conjugated with the label particles, and then to another monoclonal antibody, immobilized on an insoluble carrier. When conjugated antibodies come in contact with PAMG-1 on the Pad region, they “catch” PAMG-1 and transport it to the test region. Test region of the test strip has antibodies immobilized on it. These antibodies “meet” PAMG-1 bound to conjugated antibodies flowing up from the Pad Region. This “meeting” immobilizes the system of PAMG-1/conjugated antibodies, resulting in a brown/yellow test line that becomes visible in the test region. This line is produced by gold dye attached to conjugated antibodies and indicates a Rupture Of [fetal] Membranes. The second control line is designed to indicate that the test is functioning well. This line appears when rabbit antimouse IgG antibody ‘catches’ the mouse antibody with gold dye. Gold dye gives the resulting line its color.

EQUIPMENT AND MATERIALS:

Materials Provided: AmniSure test strips (lateral flow devices), each in foil pouch with desiccant. Each Test Strip contains:

1. AmniSure test strips (lateral flow devices), each in foil pouch with desiccant. Each Test Strip contains:
   1. Monoclonal antibody (produced by mousehybridoma)
   2. Immobilized Monoclonal antibody(produced by a different mouse hybridoma)
   3. Colloidal gold particles linked to monoclonal antibody.
   4. Mouse IgG labeled by colloidal gold particles
   5. Rabbit antimouse IgG antibody
2. Sterile Polyester vaginal swabs
3. Plastic vials with water solvent. Solvent solution contains:
   1. 0.9% NaCl
   2. 0.1% Triton X100
   3. 0.01% NaN3

SPECIMENS:

Sample from the surface of the vagina. Use the sterile polyester swab provided.

IMPLEMENTATION: TEST PROCEDURE

1. Take the solvent vial by its cap and shake well to make sure all liquid in the vial has dropped on the bottom. Open the solvent vial and put it in a vertical position.
2. To collect a sample from the surface of the vagina use the sterile polyester swab provided. Remove the sterile swab from its package following instructions on the package. The polyester tip should not touch anything prior to its insertion into vagina. Hold the swab in the middle of the stick and, while the patient is lying on her back, carefully insert the polyester tip of the swab into the vagina until the fingers contact the skin no more than 2-3 inches (5-7 cm) deep. Withdraw the swab from the vagina after one minute.
3. Place the polyester tip into the vial and rinse the swab in the solvent by rotating for one minute.
4. Remove and dispose of the swab.
5. Tear open the foil pouch at the tear notches and remove the AmniSure® test strip.
6. Dip the white end of the test strip (marked with arrows) into the vial with solvent for no less than 5 minutes and no longer than 10 minutes. Strong leakage of amniotic fluid will make the results visible early while a very small leak will take the full 10 minutes. Positive results can be read as soon as two lines appear in the test region, which sometimes occurs within seconds. Negative results will still require a full ten minutes in order to ensure accurate interpretation.
7. Remove the test strip if two stripes are clearly visible in the vial or after 10 minutes sharp. Read the results by placing the test on a clean, dry, flat surface. Do not read or interpret the results after 15 minutes have passed since dipping the test strip into the vial.
8. The darkness of the stripes may vary. The test is valid even if the stripes are faint or uneven. Do not try to interpret the test result based on the darkness of the stripes
9. If only a control line is visible, the test result is negative. If both control and test lines are visible, the test result is positive. If no lines are visible, the test result is invalid.

1	2	3
Use the sterile polyester swab provided	Rinse the swab in the solvent by rotating for one minute	Dip the white end of the test strip into the vial for no less than 5 minutes and no longer than 10 minutes.

VI. INTERPRETATION OF RESULTS:.

ONE LINE: NO MEMBRANE RUPTURE
TWO LINES: MEMBRANE RUPTURE
NO LINE:  TEST IS INVALID

EXPECTED VALUES

Leakage of amniotic fluid is indicative of the fetal membranes rupture in all women. Concentration of PAMG-1 in cervical and vaginal secretions of pregnant women without complications in pregnancy is ranged from 0.05 to 0.22 ng/ml.

When vaginitis or non-significant admixture of blood serum is present, the background level of PAMG-1 can reach the maximum of 3ng/ml. PAMG-1 concentrations in the amniotic fluid fall into 2,000-25,000 ng/ml range.

Clinically significant leakage of amniotic fluid increases  PAMG-1 concentration in cervico-vaginal secretions by a factor of thousands. The sensitivity threshold of the AmniSure Test is set by a factor of 20 above the background level of PAMG-1 (AmniSure detects 5-7 ng/ml of PAMG-1).

PRECAUTIONS AND WARNINGS

1. AmniSure test kit is for in vitro diagnostic use only. Do not take any part of the test internally.
2. A false-negative test may result in an inadequate level of care for newborns less than 37 weeks gestation if device is used in institutions other than those equipped to care for preterm infants (e.g. Level II-III nurseries). False negative results can delay the diagnosis of rupture of membranes and can increase the risk of chorioamnionitis, oligohydramnios and fetal umbilical cord accident. Negative results alone may not rule-out membrane rupture. The performance of the AmniSure test has not been established in the presence of meconium in the amniotic fluid.
3. Read and follow exactly the directions for use. Failure to do so may result in inaccurate results.
4. Safety precautions should be observed when collecting, handling, and disposing of test samples. Do not use damaged components of the test.
5. Used test kits are biohazardous. Take proper precautions when handling/discarding used test kits.
6. Do not use after the “Use By” date, which is printed on the foil pouch and on the box labeling.
7. Do not reuse the test kit components.
8. Do not bend or fold the test strip (dipstick) or the aluminum foil pouch with the Test strip in it.
9. Interrupted leakage with minimal residual fluid can lead to false negative result.
10. Presence of blood, collected with swab, can lead to false positive result.
11. Until the diagnosis of membrane rupture is excluded, avoid digital cervical examination to prevent infection and shorten the latency period.

STORAGE AND STABILITY

Store the kit in a dry place at 4 to 24oC (40 to 75oF). DO NOT FREEZE. When stored in the foil pouch at the recommended temperature, the test is stable until the “Use By” date on the foil pouch. AmniSure test should be used within six (6) hours after removing from foil pouch.

QUALITY CONTROL:

1. INTERNAL CONTROL:
   1. Each AmniSure test has built-in reagent and procedural controls to assure accurate reading of the results. The appearance of one or two lines in the test results area verifies the integrity of the test procedure.
   2. The appearance of the control line assures that adequate sample volume was present and that adequate capillary migration (lateral flow) of the sample has occurred.  It also verifies proper assembly of the test strip by manufacturer.
2. EXTERNAL CONTROL:
   1. Freeze-dried PAMG-1 protein is used for positive external control. Distilled water is used for negative external control. External controls are done for every new amisure kit lot. Point-of-Care-Testing Coordinator will perform external control testing on all new lots before kit is placed in service.  External controls will be run whenever there is suspicion that product performance is compromised, or whenever kits have not been stored according to its labeling instructions. Notify Point-of-Care-Testing Coordinator at St. Peter’s Hospital Laboratory if kit integrity is question. External controls will be provided, and/or performed by Point-of-Care-Testing.

QUALITY CONTROL PROCEDURE:

1. External Positive Control:  Fresh human amniotic fluid
2. External Negative Control: Saline solution.
3. After dissolving Freeze-dried PAMG-1 protein (amniotic fluid) with the saline solution (or distilled water), the obtained solution can be stored under refrigeration at 4-8°C for up to 24 hours. It is preferred, however, to run the QC procedure immediately after preparing a sample of diluted amniotic fluid.
4. In a glass test tube containing 0.1 ml of human amniotic fluid, add 4.9 ml of saline solution.  Mix for a few seconds.
5. Take 0.25 ml of amniotic fluid and saline solution and transfer to a small glass tube containing 0.75 mLs of saline solution to obtain a 1.0 ml solution with minimal PAMG-1 concentration.  Mix.
6. Transfer the solution to a vial similar to the vial supplied in the AmniSure test kit.
7. Dip the white end of the test strip into the vial with solvent for exactly 10 minutes. 
8. Remove the test strip after exactly 10 minutes.
9. Read results by placing the test strip on a clean, dry, flat surface. 
10. Do not interpret results after 15 minutes have passed since dipping test strip into vial.

1. LIMITS OF THE TEST

1. The AmniSure [fetal] Membranes Rupture Test is for the in vitro detection of human amniotic fluid PAMG-1 protein in vaginal secretion of pregnant woman. The test should be used to evaluate patients with clinical signs/symptoms suggestive of fetal membranes rupture.
2. You must follow all directions carefully to get an accurate reading of the results
3. Each test is a single use disposable unit and cannot be reused.
4. The AmniSure [fetal] Membranes Rupture Test results are qualitative.
5. No quantitative interpretation should be made based on the test results.
6. Presence of significant blood, collected with the swab, can lead to false positive result. In cases of only trace amounts of blood on the swab the test functions properly.
7. In very rare cases when a sample is taken 12 hours or later after a rupture, a false negative result may occur due to obstruction of the rupture by fetus or resealing of the amniotic sac
8. AmniSure® should not be used earlier than 6 hours after the removal of any disinfectant solutions or medicines from the vagina.
9. Test performance in patients without signs or symptoms of ROM is unknown
10. Results should be used in conjunction with other clinical information.
11. Bleeding, placenta previa, and performing digital exams prior to sample collection can lead to inaccurate test results.
12. Failure to detect membrane rupture does not assure the absence of membrane rupture.
13. Women may labor spontaneously despite a negative test result.
14. False negative results and delay in the diagnosis of rupture of membranes can increase the risk of chorioamnionitis, oligohydramnios and fetal umbilical cord accident.
15. The performance of the AmniSure Test has not been established in the presence of the following contaminants: meconium, anti-fungal creams or suppositories, K-Y Jelly, Monistat, Baby Powder (Starch and Talc), Replens, Baby Oil
16. Reasons for test failure:
    1. Failure to follow test instructions
    2. Improper storage of test kit.
    3. Test result read after 15 minutes are passed after the Test strip is dipped into the vial

2. INTERFERENCE

1. AmniSure’s sensitivity level is 5 ng/ml, excluding any interferences resulting from infections, sperm and urine.
2. Monoclonal antibodies used in AmniSure doesn’t cross-react to other proteins at the sensitivity of AmniSure test to PAMG-1.

DOCUMENTATION:

1. Document the internal procedural control, the patient’s test result, date and time of testing and operators initials on the log sheet.
2. All AmniSure patient test results are documented in Meditech as well as the internal QC result.

1. Excerpt from “Technical Innovations in Clinical Obstetrics,” Joong Shin Park and Errol Norwitz. Contemporary OB/GYN, September 15, 2005, vol. 50.
2. Cousins LM et al., “AmniSure Placental Alpha Microglobulin-1 Rapid Immunoassay versus Standard Diagnostic Methods for Detection of Rupture of Membranes”, American Journal of Perinatology, Volume 22, 2005
3. AmniSure ROM test package insert 11/20/07
4. Manufacture-recommended QC Reference Sheet and Procedure Manual

PLACED INTO SERVICE:  2/20/09