TITLE: RAPID FETAL FIBRONECTIN ASSAY ON TLiIQ SYSTEM

PRINCIPLE:

Fetal Fibronectin is a basement membrane protein produced by the fetal membranes that functions as an adhesion binder of the placenta and fetal membranes to the maternal decidua. In normal pregnancy, fFN is present in cervical secretions until about 16-22 weeks and is absent (negative) from about 22-35 weeks. Positive results during this time, reflecting the presence of fFN, are predictive of preterm delivery. fFN testing has been approved for risk determination in both symptomatic and asymptomatic patients.

In symptomatic patients: a negative fFN test “rules out” preterm delivery (<1% will deliver within the next 14 days); a positive fFN test allows intervention for those with the highest risk of preterm delivery in the next 14 days.

The Rapid Fetal Fibronectin assay is a qualitative test for the detection of fFN and consists of the Rapid fFN Cassette used in the TLiIQ Analyzer. The Rapid fFN Cassette is a lateral flow, solid-phase immunosorbent device designed to qualitatively detect fFN in cervicovaginal secretions collected with the Hologic Specimen Collection Kit. The cervicovaginal specimen is extracted into a buffer, warmed, and filtered with a plunger filter. Filtered sample (200 L) is dispensed onto the sample application well of the Rapid fFN Cassette. The sample flows from an absorbent pad across a nitrocellulose membrane via capillary action through a reaction zone containing murine monoclonal anti-fFN antibody conjugated to blue microspheres (conjugate); the monoclonal antibody is FDC-6, specific for fFN. The conjugate, embedded in the membrane, is mobilized by the flow of the sample. The sample then flows through a zone containing goat polyclonal anti-human fibronectin antibody which captures the fibronectin-conjugate complexes. The remaining sample flows through a zone containing goat polyclonal anti-mouse IgG antibody which captures unbound conjugate, resulting in a control line. After 20 minutes of reaction time, the intensities of the test line and control line are interpreted with the TLiIQ Analyzer.

INTENDED USE (CLINICAL REASONS FOR TESTS)

The Rapid fFN assay is a device to be used as an aid in assessing the risk of preterm delivery in less than or equal to 7 or 14 days from the time of vaginal sample collection in pregnant women with signs and symptoms of early preterm labor, intact amniotic membranes and minimal cervical dilatation (<3 cm), sampled between 24 weeks, 0 days and 34 weeks, 6 days gestation.

The device is further indicated for use in conjunction with other clinical information as an aid in assessing the risk of preterm delivery in less than or equal to 34 weeks, 6 days when a cervicovaginal sample is obtained during a routine prenatal visit between 22 weeks, 0 days and 30 weeks, 6 days of gestation in women with a singleton gestation.

SPECIMEN COLLECTION/TREATMENT:

Specimen Collection

a.	Obtain the specimen using the Hologic Specimen Collection Kit.

Collection from Symptomatic Women:

The specimen should be obtained from the posterior fornix of the vagina during a sterile speculum examination. The Dacron swab provided in the Specimen Collection Kit should be inserted into the vagina and lightly rotated across the posterior fornix for approximately 10 seconds to absorb the cervicovaginal secretions.

Collection from Asymptomatic Women:

i.	The specimen can be obtained from either the posterior fornix of the vagina or the ectocervical region of the external cervical os during a sterile speculum examination.

ii.	The Dacron swab provided in the Specimen Collection Kit should be inserted into the vagina and lightly rotated across the posterior fornix or around the ectocervical region of the external cervical os for approximately 10 seconds to absorb the cervicovaginal secretions.

b.	Once the specimen is obtained, carefully remove the swab from the vagina or cervical os and place it into the tube of buffer provided with the Specimen Collection Kit.

c.	Break the shaft (at the score) even, with the top of the tube. Align the shaft with the hole inside the tube cap and push down tightly over the shaft, sealing the tube.

Label the Specimen Transport Tube with the patient’s name and demographic information using the Laboratory barcodes or the chart labels.

Specimen Transport and Storage

Transport Specimens

Transport specimens at 2C to 25C. It is ideal to transport specimens at 2C to 8C; however, the integrity of the specimen is maintained at temperatures up to 25C for 8 hours.

b.	Exposure of the specimen to heat for even short periods of time can be detrimental. It is best to keep the specimen cool whenever possible.

Specimen Storage

Specimens must be stored refrigerated at 2C to 8C and assayed within 3 days of

collection to avoid degradation of fFN.

Store in refrigerator, if testing is delayed.

Avoid extreme temperatures.

Discard specimens after testing in labeled biohazard containers.

3.	Unacceptable Specimens

Specimens collected in or by any sample device other than the Hologic

Specimen Collection Kit.

Specimens with insufficient volume for testing or without swab.

Specimens received unlabeled.

Specimens received >3 days after the sampling date.

Specimens received at temperatures >25 C.

CALL FLOOR OR PHYSICIAN IN CHARGE OF PATIENT. NOTIFY HIM/HER OF REASON FOR

REJECTION AND REQUEST ANOTHER SPECIMEN. DOCUMENT IN LIS BY ATTACHING A

CANNED MESSAGE FOR RECOLLECTION THEN PUT THE ORDER IN FOR RECOLLECTION.

REAGENTS AND MATRIALS NEEDED:

TLiIQ System, Catalog No.: 01202. TLiIQ and Printer should be operated at room temperature (18 to 30C / 64 to 86F).

TLiIQ Analyzer

Printer

User Manual

TLiIQ QCette

Rapid fFN for the TLiIQ system, Catalog No.: 01200. Stored at room temperature (15 to 30C / 59 to 86F).

Rapid fFN Cassettes:

26 cassettes containing all necessary reagents (mouse monoclonal anti-fFN antibody conjugated to blue microspheres, goat polyclonal anti-human fibronectin antibody, and goat polyclonal anti-mouse IgG antibody) dried onto nitrocellulose membranes. Each cassette contains a desiccant and is sealed in a foil pouch.

Stability

The shelf life of the Rapid fFN Cassette is 18 months from the date of manufacture.

Unopened cassettes may be used until the expiration date printed on the foil pouch.

Once the foil pouch is opened, the Rapid fFN Cassette should be used immediately.

The Rapid fFN Cassette should be stored at room temperature (15 to 30 C / 59 to 86F).

Rapid fFN Control Kit, Catalog No.: 01166. Store in Coag tall one

door refrigerator at 2 to 8C.

Contains: One Rapid fFN Positive Control: 1.2 mL (0.900 – 01.00 ug/mL.

One Rapid fFN Negative Control: 1.2 mL (0.0.0 ug/mL

The shelf life of the Rapid fFN Calibration/Control Kit is one year from manufacture.

Unopened Controls may be used until the expiration date printed on the bottle.

Once opened, they should be used within 6 months. However, they should not be used if they are cloudy or discolored.

Tliq QCette, Catalog No.: 01175. Stored at room temperature (15 to 30/59 to 86F).

Contains:	One TLiIQ QCette

Directional Insert: One

5. One 200 uL pipettor with tips

QUALITY CONTROL:

Daily quality control of the TLiIQ analyzer is run once every 24 hours and whenever technical service requests that quality control of the TLiIQ analyzer is performed. The TLiIQ QCette is the device for use in monitoring the performance of the TLiIQ analyzer.

2.	Liquid positive and negative external controls, contained within the Rapid fFN Control Kit box, will be performed each time a new lot number or shipment of that lot number of Rapid fFN cassettes are to be used for testing purposes.

Internal controls are part of the TLiIQ System and are performed automatically with every test. These internal controls check for (1) a threshold level of signal at the procedural control position, (2) proper sample flow across the Rapid fFN Cassette, (3) absence of conjugate aggregation (Cassette: Pass / Fail), and (4) proper function of analyzer hardware (Analyzer: Pass / Fail). Daily QC is recorded in the LIS in Query with every patient that is run. It is not a reportable test to the HIS.

4.	New Lots: All new lots of reagents/cassettes are verified for quality and consistency of performance thru the use of both a positive and negative quality control sample run and recorded in the QC files along with the new lot calibration.

Daily Analyzer Quality Control Procedure

Reminder: Daily quality control of the TLiIQ analyzer is performed every 24 hours.

On TLiIQ Analyzer, go to fFN MAIN MENU:

Select or press 3 – DAILY QC

Enter USER ID (your initials or tech code) and press ENTER

Reminder: When entering a letter, press the number key associated with the letter, then press the arrow ( or ) key until the correct letter is displayed.

Enter the QCette SN (serial number located on green QCette), and press ENTER

If QCette SN is already present, press ENTER to confirm

Remove the QCette (green) from the black case and insert the QCette and press ENTER

Note: TLiIQ Analyzer will perform daily QC for 3-4 minutes, when complete, the result will

be displayed on the analyzer screen and the printed label as SYSTEM PASS.

A FAIL or INVALID result should be repeated.

*** Do not touch or wipe off the QCette. Tap to remove dust. Make sure strip is

clean and not separated from the QCette. Canned air can be used to get off any

dust particle also.

Place the label in laboratory fFN log

Press ESC to return to the fFN MAIN MENU

Any erratic or drastic temperature fluctuations will require the liquid controls to be run to validate the test cassettes are functioning as expected.

SETTING CALIBRATION FOR A RAPID fFN CASSETTE LOT

Reminder: Setting calibration must be performed each time a new lot of Rapid fFN cassettes are to be utilized for testing purposes. Setting calibration coordinates the calibration code with that particular cassette lot number.

When opening a new lot number, you cannot just change the Lot # and run QC. You need to “Set Calibration” with the new lot number and calibration code on the box.

“Daily QC” will flash. Daily QC is required even if it is less than 24 hours since the last run of QC.

Run Liquid QC. Place labels in the binder.

If there is still some of the current (old) lot left, you need to again “Set Calibration” with the old lot and again enter the old lot Calibration Code off the box.

You will need to again perform “Daily QC” as it will be flashing.

Mark all your boxes of the new lot with green dots, put the “Pass Date” and your initials on each box. The new lot is now ready for use. Log documentation of new reagent lots on the log sheet in the binder.

1.	On TLiIQ Analyzer, go to fFN MAIN MENU:

Select or press 2 – SET CALIBRATION

Enter USER ID (enter your initials or tech code) and press ENTER

Reminder: When entering a letter, press the number key associated with the letter, then press the arrow ( or ) key until the correct letter is displayed.

Enter the Adeza Cassette Lot # (located on foil pouch), and press ENTER

Enter the Calibration Code # (located on cassette box), press ENTER

TLiIQ analyzer will immediately calibrate the system with the calibration code, the result will be displayed on the analyzer screen and the printed label as SYSTEM CALIBRATED.

Place the label in the laboratory fFN log book

Press ESC to return to the fFN MAIN MENU. Daily QC will be flashing. Perform “Daily QC”.

LIQUID CONTROLS: USING INTERNAL INCUBATION MODE

POSITIVE AND NEGATIVE CONTROL PREPARATION:

Reminder: The liquid positive and negative controls are to be run each time a new lot number and or shipment of Rapid fFN cassettes are to be used for testing purposes. Run liquid QC materials with a change of new lot number.

Remove the fFN Negative Control and Positive Control vials from the Rapid fFN Control Kit box.

When first opened: label with date opened, your initials, and date of expiration (6 months from date first used or the expiration on outside of Rapid fFN Control Kit box, whichever, occurs first) must be written on the outside of the Rapid fFN Control Kit box.

Shake control vials for a few seconds.

Allow control vials to come to room temperature before testing.

Liquid Control Procedure:

Note: The positive/negative controls can be performed in any order preferred (this procedure will perform the negative control first).

After the controls have come to room temperature, information for the controls are entered in the following manner:

Go to the fFN MAIN MENU

Press or key to the third screen

Select or press 8 - LIQUID CONTROLS

Enter USER ID (your initials or tech code) and press ENTER

Reminder: When entering a letter, press the number key associated with the letter, then press the arrow ( or ) key until the correct letter is displayed.

Enter the last two numbers of the Cassette Lot#, and press ENTER

Select or press 1 - NEGATIVE CONTROL

Enter the Neg Control Lot # and press ENTER

The analyzer will then prompt you to insert the cassette and press ENTER

Remove a cassette from foil pouch and insert the cassette and press ENTER

The instrument will display while beeping: “ADD SAMPLE AND IMMEDIATELY PRESS ENTER”

Add 200 L of prepared negative control sample into sample well and press ENTER

Note:	The TLiIQ will automatically start a 20 minute timer which will allow the immunoassay reaction to take place, after the 20 minute internal incubation the TLi will analyze the Rapid fFN cassette for 3.5 minutes.

Set a timer for 25 minutes, so as to know when the negative control is finished and when to perform the positive control. Walk away, results will be displayed and printed in 24 minutes.

The result for the fFN Negative Control will be displayed on the analyzer display screen as PASS OR FAIL. The analyzer will also automatically print the result.

Place the Negative Control sticker on the daily fFN log sheet.

Press ESC to return to the fFN MAIN MENU

Repeat steps a through k, for the Positive Control.

Expected Results:

1. Negative Control:

Results: PASS.

2. Positive Control:

Results: PASS.

Out of Control Procedure:

Repeat test

Refer to Users Manual for details and troubleshooting

Call Hologic Technical Support at 1-877-945-0208. If they do not respond in a timely

manner call your Hologic Territory Representative/Manager.

Notify lead technologist and/or manager

Recording QC Results:

Record on QC Worksheet: Lot # of controls, expiration dates, result of PASS or FAIL, date, and your tech initials, along with any comments if needed.

PATIENT SAMPLE TESTING: USING INTERNAL INCUBATION MODE

Patient Sample Preparation:

Upon receipt of bar coded or labeled patient sample in Specimen Transport Tube, invert tube several times to prevent aggregation of fFN.

Allow patient Specimen Transport Tube to come to room temperature before testing.

Mix Specimen Transport Tube by flicking the bottom of the Specimen Transport Tube several times while holding the top of the tube firmly.

Sample is now ready for testing.

Patient Sample Testing Procedure:

From the fFN MAIN MENU, select or press 1 - TEST PATIENT

Enter USER ID (your initials or tech code) and press ENTER.

Reminder: When entering a letter, press the number key associated with the letter, then press the arrow ( or ) key until the correct letter is displayed.

Enter the last two numbers of the Hologic Cassette Lot #, and press ENTER.

Enter patient Order Number and press ENTER.

Insert the patient cassette and press ENTER.

The instrument will display: “ADD SAMPLE AND IMMEDIATELY PRESS ENTER”

Add 200 L of prepared patient sample into sample well and press ENTER.

The analyzer will begin a 20-minute incubation countdown. The screen will display:

TEST IN PROCESS

DO NOT REMOVE

CASSETTE

19 MIN 59 SEC

Following the 20 minute incubation, the analyzer will begin the analysis of the cassette. The screen will display:

TEST IN PROCESS

ANALYZING

DO NOT REMOVE

CASSETTE

When the test is complete, the system will automatically display and print the result.

For Example:

RAPID fFN RESULT

NEGATIVE

TIME: 01:30AM DATE: 7/7/00

--------------------------------------

PATIENT: 1234

USER: KGG

CASSETTE LOT: L0051

CALIBRATION CODE: EC42

ANALYZER ID: 00812

--------------------------------------

INTERNAL CONTROLS

ANALYZER: PASS

CASSETTE: PASS

Place the printed fFN result in the laboratory fFN log book.

Press the ESC key to return to the fFN MAIN MENU.

REPORTING RESULTS:

Entering Patient Results into the LIS:

1. Enter patient results in LIS:

Function: Resulting Worklist: COAG

b.	Type in your order number

c.	Choose your result from the keypad. Enter result as POSITIVE, NEGATIVE or INVALID.

d.	Enter any canned message as necessary. There are 2 associated with FFN’s. One for "bloody" specimens, and, another for "invalid" results. Add canned message @BLDY on any bloody sample. Add @INVL for an Invalid result. (See examples following this policy).

e. Verify your results.

2.	Attach QC/ and Patient result printout on Daily Log Sheets.

Interpretation of Results:

Positive Result:

The sample result is positive if the patient sample is greater than or equal to 50 g / mL

fFN, which is determined by the factory set calibration for that particular cassette lot number.

Negative Result:

The sample result is negative if the patient sample is less than 50 g / mL fFN, which is determined by the factory set calibration for that particular cassette lot number.

Invalid Result or Error Message:

Invalid Result

If you should obtain an invalid test result, note the sample appearance:

NOTE: If specimen is invalid and appears viscous, centrifuge specimen at 2500 rpm for 5 minutes, then retest using 200L of supernatant fluid (Cancel, with QNS if less than 200L supernatant is present to repeat.)

Retest with 200 L of additional patient sample on a new cassette.

If repeat test is invalid, report test results in LIS system as INVALID. Place invalid cassettes and copy (press print button once result is displayed) of invalid printer results by the analyzer for inspection by tech and Sr. Tech.

If invalid results occur, analyze the invalid cassettes. The following are reasons why invalid results may occur:

if there is a clean white membrane with no blue lines present, the fFN sample was probably not added to the cassette or there was a very thick sample which inhibited sample flow.

if there is no control line (blue line furthest from sample well), then the sample flow was not sufficient, giving an invalid result. The control line is an internal QC to assure that there is sufficient sample flow to give a valid patient line. This most likely is due to mucous or other material (jelly to lubricate sterile speculum) in the sample that inhibits the flow across the nitrocellulose membrane.

if the patient line is heavy dark blue and the control line has a very faint or no blue color, this typically indicates the fFN sample submitted contained amniotic fluid from either ruptured membranes or a small leak in the amniotic sac. Amniotic fluid is a contraindication to the test, because it contains large amounts of fetal fibronectin. Therefore, the patient line was saturated with antibodies (containing the blue conjugate) so much as to not leave any for the control line, which the instrument interprets as a flow problem with the sample.

if the membrane is discolored from a bloody fFN sample, this will give an invalid result because the membrane becomes so discolored as to not allow a large enough difference from the background to the control line and the instrument will not be able to give a valid result.

Report out as invalid and add canned message: “@INVL”. “Integrity of sample may be compromised, test was repeated”, populates the screen, review and verify.

Error Messages

Turn off the TLi Analyzer, wait 10 seconds, then turn the TLi Analyzer back on and allow the instrument to go through diagnostics test. When the fFN Main Menu is displayed, retest sample.

If problem persists, call Hologic Technical Support at 1-877-945-0208. If they do not respond in a timely manner call Hologic Territory Representative/Manager.

SYSTEM INITIALIZATION

This procedure needs only to be performed if (a) a new TLiIQ analyzer is received ( prior to running the QCette as a quality control device), (b) a replacement QCette is received, or (c) instructed to perform system initialization by Hologic customer support.

System Initialization Procedure

From the fFN MAIN MENU, press 6 – CHANGE SETUP

Select or press 4 – QCette SETUP.

Enter USER ID (your initials or tech code) and press ENTER.

Reminder: When entering a letter, press the number key associated with the letter, then press the arrow ( or ) key until the correct letter is displayed.

Enter the QCette SN (#) and press ENTER.

This serial number is located on the green QCette

Insert the QCette and press ENTER.

The instrument will display: INITIALIZING QCette DO NOT REMOVE CASSETTE

This process will take approximately 9 minutes in which the QCette Setup initializes the QCette for use in evaluating the performance of the analyzer. During the initializing process, the performance criteria of the analyzer are established.

When finished the TLiIQ screen will display QCette SETUP COMPLETE, press ESC to exit to fFN MAIN MENU.

PRECAUTIONS:

Do not use glass tubes or pipettes, as fFN binds to glass. Tubes and pipettes of polypropylene or polyethylene are acceptable.

Allowing Positive and Negative Controls and patient samples to come to room temperature before testing is necessary to ensure homogeneity.

Do not mix materials from different kit lots.

Do not use reagents beyond the expiration date printed on the kit box.

Do not use reagents that are cloudy or discolored.

Avoid cross-contamination of reagents. When using the MLA pipette, change tips between each calibrator, control and patient samples. Recap reagents tightly with the correct color-coded caps.

Source material used to prepare the Reference Calibrator and Controls is of human origin. The donors were tested and found to be negative for HIV1, HIV2, HCV antibody and hepatitis B surface antigen using established methods. No known test method can offer total assurance that HIV, hepatitis C virus, hepatitis B virus or other infectious agents are absent. Handle these reagents and all patient specimens as if potentially infectious.

LIMITATIONS OF PROCEDURE:

The Rapid fFN result should not be interpreted as absolute evidence for the presence or absence of a process that will result in delivery in less than or equal to 7 or 14 days from specimen collection in symptomatic women or delivery in less than or equal to 34 weeks, 6 days in asymptomatic women evaluated between 22 weeks, 0 days and 30 weeks, 6 days of gestation. A positive Rapid fFN result may be observed for patients who have experienced cervical disruption caused by, but not limited to, events such as sexual intercourse, digital cervical examination, or vaginal probe ultrasound. The Rapid fFN result should always be used in conjunction with information available from the clinical evaluation of the patient and other diagnostic procedures such as cervical examination, cervical microbiological culture, assessment of uterine activity, and evaluation of other risk factors.

Test results may not be interpreted visually and must be based on the use of the TLi system.

Modification of the assay protocol described herein may yield erroneous results.

The assay has been optimized with specimens taken from the posterior fornix of the vagina or the ectocervical region of the external cervical as. Samples obtained from other locations should not be used.

Assay interference from the following components has not been ruled out: douches, white blood cells, red blood cells bacteria, and bilirubin.

Manipulation of the cervix may lead to false positive results. Specimens should be obtained prior to digital examination or manipulation of the cervix.

Care must be taken not to contaminate the swab or cervicovaginal secretions with lubricants, soaps, or disinfectants. These substances may interfere with absorption of the specimen by the swab or with the antibody-antigen reaction of the assay.

Test results are difficult to interpret if the specimen contains semen or if the specimen was collected less than 24 hours after coitus. Studies have shown that the presence of semen may lead to a false positive result. (20-50% chance of false positives).

Patients with suspected or known placental abruption, placenta previa, or moderate or gross vaginal bleeding, ruptured membrane should not be tested for fFN.

10.	The test should not be used with advanced cervical dilation (>3cm).

CANCEL TEST, WHILE TESTING STILL IN PROGRESS:

To cancel a test while still in progress, and results have not yet been printed, press ESC twice and respond to screen request.

MAINTENANCE:

As Needed:

Clean dust off of TLiIQ system as needed.

Clean off the QCette with canned air or by tapping it on the counter with QCette on its side.

REFERENCES:

1. Adeza User’s Manual, 2001

2. Rapid fFN Cassette Package Insert 4/2001

3. Most recent CAP checklist.

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