Title: D-DIMER (FEU) Method by Innovance for the Sysmex CA 1500

PRINCIPLE:

Coagulation activation results in the cleavage of fibrinogen to fibrin. The fibrin monomers spontaneously aggregate to fibrin and are cross-linked by Factor XIII; this produces a fibrin clot. In response to the coagulation process the fibrinolytic system is activated resulting in the conversion of plasminogen into plasmin, which cleaves fibrin (and fibrinogen) into the fragments D and E. Due to the cross-linkages between the D-domains in the fibrin clot, the action of plasmin releases fibrin degradation products with cross-linked D-domains. The smallest unit is the D-Dimer. Detection of D-Dimers, which specifies cross-linked fibrin degradation products generated by reactive fibrinolysis, is an indicator of coagulation activity.

CLINICAL SIGNIFICANCE:

Elevated D-dimer levels are observed in all diseases and conditions with increased coagulation activation, e.g. thromboembolic disease, disseminated intravascular coagulopathy (DIC), acute aortic dissection, myocardial infarction, malignant diseases, obstetrical complications, third trimester of pregnancy, surgery or polytrauma.1-6

The relevance of the D-Dimer assay is as an aid in the diagnosis of thromboembolic events. Elevated concentrations of D-Dimer are indicative of the presence of a clot and have been reported in deep vein thrombosis, pulmonary embolism and disseminated intravascular coagulation. Innovance D-Dimer assays have been FDA cleared to exclude both PE and DVT as of January 2011.

This assay is intended for use in conjunction with a non-high clinical pre-test probability (PTP) assessment model to exclude deep vein thrombosis (DVT) and pulmonary embolism (PE).

Polystyrene particles covalently coated with monoclonal antibody (8D3)8 are aggregated when mixed with samples containing D-Dimer. D-Dimer cross-linkage region has a stereosymmetrical structure, i.e. the epitope for the monoclonal occurs twice. Consequently, one antibody suffices in order to trigger and aggregation reaction, which is then detected turbimetrically via the increase in turbidity.

SPECIMEN COLLECTION:

Type:

Mix nine parts of freshly collected blood with one part of 0.11mol/L (3.2%) sodium citrate anticoagulant, avoiding the formation of foam. Invert the tube gently three or four times immediately after venipuncture to ensure proper mixing of blood and anticoagulant. A syringe or evacuated tubes (blue top) may be used with caution for collection. If multiple specimens are collected, the coagulation sample should be the first or second tube collected. If blood is drawn from an indwelling catheter, the line should be flushed with 5.0 mL saline and the first 5.0 mL of blood or six dead space volumes of the catheter discarded or used for other laboratory tests. The citrate concentration must be adjusted in patients who have hematocrit values above 55%. Specimens that are clotted, collected in the wrong tube, have visible hemolysis (greater than slightly hemolyzed) or have less than the expected fill should be rejected.

Handling Conditions:

The specimen should be transported at room temperature. The whole blood specimen is checked for clot formation by gentle inversion and checked with applicator sticks, then centrifuged and tested within 4 hours from the time of specimen collection. Centrifuge the capped specimen tube for a minimum of 15 minutes at 1500 g to consistently produce platelet poor plasma (platelet count

<10 x 109/L). If immediate testing is to be done, the plasma may remain on the packed cells or separated. To separate plasma, use a plastic transfer pipette; remove the plasma to a plastic tube. If testing is not completed within 4 hours or if shipment is required, the plasma may be stored frozen at -18 C or below for up to 4 weeks if frozen within 4 hours of blood collection. Frozen plasma samples must be rapidly thawed at 37 C while gently mixing and tested within 2 hours. Do not refreeze.

REAGENTS AND EQUIPMENT:

Equipment:

Sysmex CA-1500 Instrument

4.0 mL Sample Cups

Reaction Tubes

Sample Plates

Waste and Rinse Bottles

Materials:

Innovance D-Dimer Kit containing:

Innovance D-Dimer Reagent
Innovance D-Dimer Buffer
Innovance D-Dimer Supplement

Innovance D-Dimer Diluent

Innovance D-Dimer Calibrator

Control Material:

Innovance D-Dimer Control 1
Innovance D-Dimer Control 2

CA Clean I

Preservative-free distilled or deionized water

Preparation:

All components of a kit are lot-specific. The combination of lots other than those specified for the particular kit lot may lead to incorrect results.

Follow the preparation instructions prior to use according to Table 1. Storage instructions are detailed in section “Storage and Stability”.

Table 1: Instructions for the preparation of the kit components

Instructions	Innovance D-Dimer Reagent	Innovance D-Dimer Buffer/Supplement/Diluent	Innovance D-Dimer Calibrator
Reconstitution	Dissolve with 4.0 mL distilled water Invert 3 times Leave the vial for at least 15 minutes at 15 -25 º C	Ready to use	Dissolve with 1.0 mL distilled water Mix carefully without foam formation Leave the vial for at least 15 minutes at 15 -25 º C
Prior to placing on the system	Mix well (again) by inverting 3 times Avoid foam formation Remove bubbles	Mix carefully Avoid foam formation Buffer only: resuspend potential precipitates by gently swirling. Any residual precipitates after resuspension do not impact test results Remove bubbles	Mix (again) careful Do not use if vial contains visible clot. 3. Label the vial with the open date/time it was reconstituted and the 4 hour expiration time.
Aliquoting	1. Mix well (again) by inverting 3 times 2. Aliquot into an empty vial provided with the same kit 3. Discard empty vials if unused until complete consumption of the kit.	1. 1. Aliquot into an empty vial provided with the same kit. 2. Discard empty vials if unused until complete consumption of the kit.	N/A
Freeze and thaw	Use the original container or the empty vial provided with the same kit Follow storage instructions in the section “Storage and Stability: Thaw at 37 °C within 10 minutes. Thereafter the vial may no longer be stored at 2 – 8 °C. Do not freeze again after thawing.	Refer to section “Storage and Stability”
Placing on the system	Place Innovance D- Dimer Reagent in Reagent Holder position C 1.	Place Innovance D-Dimer Buffer in Reagent Holder positions B 6. Place Innovance D-Dimer Supplement in Reagent Holder positions B 8. Place Innovance D-Dimer Diluent in Reagent Holder positions D 10	Place Innovance D-Dimer Calibrator in Reagent Holder positions D1-D14 or Sample Rack position 1.
Note	The reconstitution, opening, or freezing date may be noted on the vial label using the framed free space.

Storage and Stability:

The kit may be used up to the expiry date indicated on the label if stored unopened at 2 – 8 °C.

Table 2: Stability after reconstitution or first opening (closed vial)

Temperature	Innovance D-Dimer Reagent	Innovance D-Dimer Buffer	Innovance D-Dimer Supplement	Innovance D-Dimer Diluent	Innovance D-Dimer Calibrator
2 -8 ° C	4 weeks	4 weeks	4 weeks	4 weeks	-
≤ -18 °C	4 weeks	4 weeks	4 weeks	4 weeks	-
15 – 25 °C	-	-	-	-	4 hours
Onboard stability	24 Hours	24 Hours	24 Hours	24 Hours

Do not refreeze and thaw. Follow freeze and thaw instructions in section “Preparation of the Reagents”.

Innovance D-Dimer Control 1:
Innovance D-Dimer Control 2:
Lyophilized human plasma based products containing D-Dimer containing

`	5-chloro-2-methyl-4-isothiazole-3-one

2-methyl-4-isothiazole-3-one (< 1 mg/L)

sodium azide (< 1 g/L)

Assayed controls used for the assessment of precision and analytical bias in the normal and pathological range for the determination of D-Dimer in the Innovance D-Dimer Kit.

Reconstitute the Innovance D-Dimer Control 1and 2 with 1.0 mL of deionized water.

Re-stopper vial and invert gently to dissolve (without foam formation).

Let stand at +15 to +25 °C for at least 15 minutes before use

Mix carefully once before using

Stability after reconstitution:

-18 C:	4 weeks (do not refreeze)**
2- 8 C:	7 days (closed vial)
15-25 C:	8 hours (closed vial)

**Must be frozen in the original containers. Do not refreeze after thawing.

Innovance D-Dimer Controls 1 and 2 may be frozen in the original contained and thawed once after reconstitution. The previous storage time at 15-25 C must not have exceeded 4 hours. The plasma must be well sealed and frozen as quickly as possible. Thawing must be completed at 37 C and within a maximum of 10 minutes. Innovance D-Dimer Control 1 and Innovance D-Dimer Control 2 should not stand for more than 4 hours at 15-25 C after thawing. Innovance D-Dimer Control 1 and Innovance D-Dimer Control 2 should not be used if they contain visible clots.

CA Clean I is liquid and ready for use

Store at 2-8 °C. Do Not Freeze.

Stable unopened at 2-8 °C until expiration date on bottle.

Opened bottle stable for 30 days at 2-8 °C.

These products are for in vitro diagnostic use only.

WARNING: Each donor or donor unit was tested and found to be negative for human immunodeficiency virus (HIV) 1 and 2, hepatitis B virus (HBV) and hepatitis C virus (HCV) using either tests found to be in conformance with the In Vitro Diagnostic Directive in the EU or FDA approved tests. Because no known test can offer complete assurance of the absence of infectious agents, all human derived products should be handled with appropriate caution.

WARNING: Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. If discarded into a sink, flush with a large volume of water to prevent azide build up.

Reagent Integrity:

Indication of deterioration: No evidence of vacuum in vial upon opening, difficulty in reconstituting reagent, control values outside of determined range

QUALITY CONTROL:

Innovance D-Dimer Control 1

Innovance D-Dimer Control 2

Innovance D-Dimer Controls must be tested at least every eight hours of testing and for each vial of reagent for the respective measurement range to ensure that the system is functioning correctly. Control of the lower measurement range is performed with Innovance D-Dimer Control 1, and for the upper range with Innovance D-Dimer Control 2.

The measured values obtained must be within the acceptance ranges given in the respective Table of Assigned Values.

If values are obtained outside the acceptance ranges, the measurement must be repeated. If the deviations are confirmed, a new calibration must be performed.

Do not report patient results unless the cause of the deviating control results has been identified and corrected!

PROCEDURES FOR SETUP:

Loading Reagents
(when the instrument is not performing a test)

Press the [Set Reagents] key from the 'Main Menu'. The 'Consumable' screen will appear.

Confirm that the lid signal (status of cover opening) is green; then open the light shield lid.

Verify the appropriate group (Group 1 or 2) is displayed on the 'Consumable' screen. The appropriate group will have the DDi reagents displayed.

Note: If the appropriate Group is not displayed, press [Select Group] to display the change Group screen. Press the correct group key, then [Return]. Verify reagent position displayed in pink on the Consumable Screen contains the correct reagent.

To add the Innovance D-Dimer Reagent, Press [DDi.REA] reagent holder position key. The reagent holder position is displayed in the upper left corner of the reagent holder position key. The position is designated with a letter (A – E) and a position number.

Place the vial of Innovance D-Dimer Reagent in the appropriate reagent holder position (C 1).

Verify that the correct lot number is displayed. To change the lot number, press [Lot #], enter the correct information and press [Enter].

Verify that the correct expiration date is displayed. To change the date, press [Exp.Date], enter the correct data in the format mm/dd/yyyy, and press [Enter].

Verify that the correct vial type is displayed. To change it press [Vial], and use the arrow keys to select the correct type and press [OK].

Press [ENTER] on the numeric keypad. The reagent holder position key for [DDi.REA] will be displayed with a green background.

Repeat procedure to add the Innovance D-Dimer Buffer [DDi.BUF] and Innovance D-Dimer Supplement [DDi.SUP] in the appropriate reagent holder position (B 6 and B 8).

Repeat the above procedure to add Innovance D-Dimer Diluent [DDi.DIL] to the reagent holder position D-10.

Repeat the above procedure to add CA Clean I reagent holder position A-2 and E-1 position, respectively.

Close the light shield lid.

Press [Return] to display the main menu

Loading Consumables and Discarding Waste Material:
(do the following as necessary)

Replenish Sample Plates

Note: Sample plate LED indicator lights show the status of the sample plate:
Green = not used, Red = partially used, Flashing Red = completely used.
On the Consumable screen, the status is indicated with colors:
Green = not used, Yellow = partially used, Red = completely used.
To replenish a Sample Plate when the instrument is not operating:

Confirm that the instrument is not operating.

Confirm that the "lid" signal (status of cover opening) is green; then open the light shield lid.

Remove the used sample plate.

Confirm on the screen that the sample plate has been replaced with a new sample plate by pressing [OK].

Place the unused sample plate on the CA 1500. The sample plates are keyed to be loaded in one direction on the CA 1500.
Caution: Do not load partially used sample plates. The instrument will not recognize partially used wells.

Close the light shield lid.

Replenish Reaction tubes

Reaction tubes can be replenished at any time during operation. Open the reaction tube hopper on the top of the CA 1500 by pressing the front part of the reaction tube hopper lid.

Replenish/Fill the reaction tubes until the red line inside the reaction tube hopper (Max. 300 reaction tubes).

Replenish Rinse Solution (If in Use)

Confirm that the instrument is not operating or has been interrupted due to insufficient rinse (error message "Replenish Rinse Fluid").

Remove the cap with the float switch from the rinse solution container by turning the cap counter-clockwise.
Caution: Do not touch the float switch.
Rinse the float switch thoroughly with deionized or distilled water if contact is made.

Replace the rinse solution with distilled or deionized water.

Replace the float switch by tightening the cap clockwise to close.

Verify that the tubing is securely connected and is not kinked.

If the analysis was interrupted due to insufficient rinse, press [Resume] key.

Dispose of Liquid Waste

Confirm that the instrument is not operating or has been interrupted due to waste is full.

Remove the cap with the float switch from the waste container by turning the cap counter-clockwise.
Caution: Biohazard

Dispose of the biohazardous liquid waste.

Replace the float switch by tightening the cap clockwise to close.

Verify that the tubing is securely connected and is not kinked.

Dispose of Used Reaction Tubes from the Trash Box

Confirm that the instrument is not operating.

On the left side of the instrument, pull out the trash box.

Discard the used reaction tubes.
Caution: Biohazard

Clean the reaction tube trash box with tap water, and thoroughly wipe off moisture from the reaction tube trash box.

Restore the reaction tube trash box.

If the volume monitoring is set to monitor the number of used reaction tubes in the trash box; a message will appear asking the operator if a reset of the number of used reaction tubes is necessary. If the used reaction tubes have been discarded press [OK]. If the used reaction tubes have not been discarded, press [Cancel].

CALIBRATION/CALIBRATION VERIFICATION

Calibration criteria include: at changes of reagent lot numbers, failure to recover QC tolerance limits, after major service or maintenance on the CA1500 or at the recommendation of the manufacturer and at approximately every 6 months.

The reference curve is valid for the respective lot of the reagent employed. A new curve should be prepared with a new lot of Innovance D-Dimer using Innovance D-Dimer Calibrator provided in the same kit and if indicated by any change in analytical conditions. The calibration curve can be used as long as the assay-dependent assigned values (i.e. Innovance D-Dimer Control 1 and Innovance D-Dimer Control 2) are within the corresponding acceptance ranges.

In order to calibrate the Innovance D-Dimer assay, a calibrator must be loaded onto the CA 1500. The calibrator (Innovance D-Dimer Calibrator = DDi.CAL) can be placed in a predefined reagent holder position (procedure A) or from position 1 of a sample rack (procedure B). Follow procedure A or B given below depending on the desired placement of the calibrator. Follow procedure C to accept/update the curve and for standard curve documentation. The package insert in the kit has the assigned calibrator value listed in concentration mg/L FEU. Ex: 4.68mg/L FEU

A. Calibrating using a pre-defined Standard Reagent Holder position:

Software Set-up for calibration from a Reagent Holder position

From the 'Main Menu', press [Standard Curve] to display the 'Standard Curve' screen.

Select the Innovance D-Dimer assay: Press [DDi].

Press [Analysis Setting] to display the 'Standard Curve Analysis Setting' screen.

Verify R. Holder is displayed for the calibrator position. If not, press [Sampler/Holder] to change the position.

Verify that Auto Dil. & Analyze is displayed for the mode. If not, press [Change Mode].

Verify that Innovance D-Dimer Calibrator (DDi.CAL) is displayed as the calibrator.

If not:

Press [Select Reagent.]

Use the up [] or down [] arrow to select [DDi.CAL].

Press [OK].

Use the up [] or down [] arrow to highlight the Assay Sheet Val. position. A keypad will be displayed.

Enter the assay value of the calibrator from the Innovance D-Dimer Calibrator lot number package insert.

Press [Enter] on the key pad.

Verify that the dilution ratios are correct. They should be 1/1, 1/2, 1/4, 1/8, 1/16, 1/32.

If not:

Use the arrow keys to highlight the incorrect dilution ratio

Press [Select Dil. Rat.].

Use the arrow keys to highlight the correct dilution ratio

Press [OK].

Verify the replication for each dilution ratio is set to 2.

If not:

Use the arrow keys to highlight the incorrect replication field.

Enter in the keypad "2".

Press [Enter] on the keypad

Verify DDi.DIL is displayed in the "Buffer" field.

If not:

Highlight the buffer field.

Press [Select Reagent].

Use the arrow keys to select [DDi.DIL].

Press [Return

Press [Return] or [Quit], whichever is highlighted. The "Update Settings?" box will be displayed.

Press [OK].

Press [Main Menu].

Load Calibrator

Press the [Set Reagents] key from the 'Main Menu'. The 'Consumable' screen will appear.

Confirm that the lid signal (status of cover opening) is green; then open the light shield lid.

Verify the appropriate group (Group 1 or 2) is displayed on the 'Consumable' screen. The appropriate group will have the [DDi.CAL] reagent holder position key displayed.
Note: If the appropriate group is not displayed, press [Select Group] to display the 'Change Group' screen. Press the correct group key, then [Return].
Verify the reagent position displayed in pink on the 'Consumable' screen contains the correct reagent.

Press [DDi.CAL] reagent holder position key. The reagent holder position is displayed in the upper left corner of the reagent holder position key. The position is designated with a letter (D) and a position number.

Pour the Innovance D-Dimer Calibrator into a 4 mL analyzer cup.

Place the analyzer cup in the appropriate reagent adapter in the appropriate reagent holder position.

Verify that the correct lot number is displayed. To change the lot number, press [Lot #], enter the correct information and press [Enter].

Verify that the correct expiration date is displayed. To change the date, press [Exp.Date], enter the correct data in the format mm/dd/yyyy, and press [Enter].

Verify that the correct vial type is displayed. To change it press [Vial], and select the correct type and press [OK].

Press [ENTER] on the numeric keypad. The reagent holder position key for Innovance D Dimer Calibrator [DDi.CAL] will be displayed with a green background.

Close the light shield lid.

Press [Return] to display the Main menu.

Start / Register the calibration from a Reagent Holder position

From the Main Menu, press [Work List] to display the 'Work List' screen.

Press [Reagent Holder] to display the 'Work Load List' and use the arrow keys to select DDi.CAL.

Press [Standard Curve] to display the 'Standard Curve Parameter Settings' screen.

Press [DDi] to register the calibration. The symbol will be displayed, indicating that Innovance D-Dimer has been selected for standard curve analysis.

Press [Return] to display the 'Work Load List' screen.

Press [Start] to begin the analysis.

Press [Main Menu] to display the Main Menu screen.

Upon completion of the calibration see procedure "C. Accept/Update the Standard Curve" below.

B. Calibrating from a Sample Rack

Software set-up for calibration from a sample rack

From the Main Menu, press [Standard Curve] to display the Standard Curve screen.

Press [DDi] to display the Standard Curve screen.

Press [Analysis Setting] to display the Standard Curve Analysis Setting screen.

Verify Sampler is displayed for the calibrator position.
If not, press [Sampler/Holder] to change the position.

Verify Auto Dil. & Analyze is displayed for the mode.
If not, press [Change Mode] to toggle between the modes.

Verify that DDi.CAL is displayed as the calibrator.
If not:

Use the up [] or down [] arrow to highlight the calibrator position

Press [Select Reagent]

Use the up or down arrow to select DDi.CAL

Press [OK]

Use the up [] or down [] arrow to highlight the Assay Sheet Val. position. A keypad will be displayed.

Enter the assay value of the calibrator from the Innovance D-Dimer Calibrator lot number package insert.

Press [Enter].

Verify that the dilution ratios are correct. They should be 1/1, 1/2, 1/4, 1/8, 1/16, 1/32
If not:

Use the arrow keys to highlight the incorrect dilution ratio

Press [Select Dil. Rat.]

Use the arrow keys to highlight the correct dilution ratio

Press [OK]

Verify the replication for each dilution ratio is set to 2.
If not:

Use the arrow keys to highlight the incorrect replication field

Enter in the keypad "2"

Press Enter on the keypad

Verify DDi.DIL is displayed in the "Buffer" field.

If not:

Highlight the buffer field

Press [Select Reagent]

Use the arrow keys to select [DDi.DIL]

Press [Return

Press [Return] or [Quit], whichever is highlighted. The Update Settings? box will be displayed.

Press [OK].

Press [Main Menu].

Load Calibrator

Pour the reconstituted Innovance D-Dimer Calibrator into a 4 mL analyzer cup.

Place the analyzer cup into the first position of the sample rack.
Note: If patients or controls are to be run, these samples must be loaded into the next sample rack and programmed in the next rack Worklist.

Load the sample rack(s) in the right input tray.

Register / Start the calibration from a sample rack

From the Main Menu, press [Work List] to display the 'Work List' screen.

Press [Rack] to choose the calibrator position.

Press [Standard Curve] to display the 'Standard Curve Parameter Settings' screen.

Press [DDi] to register the calibration. The symbol "" will be displayed, indicating that Innovance D-Dimer has been selected for standard curve analysis.

Press [Return] to display the 'Standard Curve Work List' screen.

Press [Start] to begin the analysis.

Press [Main Menu] to display the 'Main Menu' screen.

Upon completion of the calibration see procedure "C. Accept/Update the Standard Curve" below.

C. Accept/Update the Standard Curve

Update the standard curve

When the standard curve analysis is complete, press [Standard Curve] at the 'Main Menu' to display the' Standard Curve' screen.

When the Update the Standard Curve? box appears, press [OK].

Press [DDi].

Press [Update].
Note: This does not update the curve; it causes the CA-1500 to display the 'Standard Curve Update’ menu.

View the new standard curve to determine the appropriate key to press:

[Continue] returns to the previous menu when decision to accept has not been made.

[Reject Both] rejects the new standard curve and no calculations are made for samples. processed behind the Standard Curve Analysis even if a previous standard curve exists.

[Update] accepts the new standard curve.

[No-update] rejects the new standard curve, recalibration can be performed if necessary.

Both levels of QC material are run to validate the curve.

Printing the standard curve

From the Main Menu press [Standard Curve]

Select the [DDi] key for Innovance D-Dimer assay.

From the 'Standard Curve' screen, press [More].

Press [Output Input] to display the 'Standard Curve Data Output' menu.

Press [GP Print] to display the Print Confirmation message window.

Press [OK] to start printing.

Press [OK] to return to the Main Menu or press [Return] when printing is complete to remain at the Standard Curve screen.

Saving the standard curve to a diskette

From the Main Menu press [Standard Curve].

Select [DDi] for the assay.

From the Innovance D-Dimer Standard Curve screen, press [More].

Press [Output Input] to display the Standard Curve Output Input menu.

Press [Save to FD] to display the Floppy Disk Confirmation message window.

Insert a 3 1/2" IBM formatted high density double sided diskette into the disk drive on the right side of the CA 1500. The diskette must be inserted with its label facing away from you.

Press [OK]. The data will be saved onto the diskette.
Note: Only one standard curve for a particular parameter can be saved on a diskette.

Remove the diskette.

Press [OK] to return to the Main Menu or press [Return] when saving complete to remain at the Standard Curve screen.

STEPWISE PROCEDURES:

Samples can be programmed and loaded on the CA 1500 using one of four different options depending whether the sample is barcoded and/or the host is connected:

Manually enter sample ID (non-barcoded) /
No automatic inquiry of tests (no host connection or the host is down)

Manually enter sample ID (non-barcoded) /
Automatic inquiry of tests (host connection is operational)

Sample ID number (barcoded sample) read by bar-code reader /
No automatic inquiry of tests (no host connection or the host is down)

Sample ID number (barcoded sample) read by bar-code reader /
Automatic inquiry of tests (host connection is operational)

Manually enter sample ID (non-barcoded) /
No automatic inquiry of tests (no host connection or the host is down)

From the Main Menu, press [Worklist]

Verify that Register Rack is displayed above the parameter keys. If not, press [Rack]

Verify that the next available rack is displayed (white background). Use the [Prev] or [Next] key to locate the next available rack.

Use the up and down arrow keys to select the desired sample rack position, press [ID No. Entry]

The numeric keypad is displayed. Enter the sample ID number.

Press the blue test key [DDi]. Continue to select test to be performed on the sample if necessary.

Press [Enter] and [Quit] to clear the numeric ID entry keypad.

Use the arrow keys to select samples for sample specific configurations:

To perform the sample in 'Micro Mode': For samples to be performed in the Micro mode press the [Micro] key for each sample. A "" symbol will be displayed on the Micro key. Samples programmed to be performed in Micro Mode will display "M" in the "opt." column in the respective worklist position on the screen.

To perform a reflex test on a sample: Press the [Reflex] key. A "" symbol will be displayed on the Reflex key. Samples programmed for reflexive testing will display "R" in the "opt." column in the respective worklist positions on the screen.
Note: Reflexive testing cannot be performed on samples running in Micro Mode.

To remove a redilution or set a redilution, repeat analysis, and/or dilution ratio (MDA) for a particular sample: Press the [Sample Specific] key. From the 'Sample Specific' screen select the assay and select the desired re-analysis method or MDA setting for the sample. Press [Return] until the respective worklist is displayed.
Note: Redilution and dilution ratio options are only available on tests in which the test protocol includes a primary dilution.

Press [Enter]. Each test selected should show the symbol "".

Load the sample in the respective sample rack position.

Repeat until all sample ID numbers and tests are entered for the respective sample rack positions.

When all samples are programmed and loaded in the respective sample rack position, press [Quit] on the numeric keypad.

With all reagents and consumables on board, place the sample rack into the right rack pool of the sampler (input tray).

Press [Start] or [Register More] (during operation) to begin processing.

Manually enter sample ID (non-barcoded) /
Automatic inquiry of tests (host connection is operational)

From the Main Menu, press [Worklist]

Verify that Register Rack is displayed above the parameter keys. If not, press [Rack]

Verify that the next available rack is displayed (white background). Use the [Prev] or [Next] key to locate the next available rack.

Use the up and down arrow keys to select the desired sample rack position, press [ID No. Entry]

The numeric keypad is displayed. Enter the sample ID number.

Press [Enter] and [Quit] to clear the numeric ID entry keypad.

Use the arrow keys to select samples for sample specific configurations:

To perform the sample in 'Micro Mode': For samples to be performed in the Micro mode press the [Micro] key for each sample. A "" symbol will be displayed on the Micro key. Samples programmed to be performed in Micro Mode will display "M" in the "opt." column in the respective worklist position on the screen.

Press [Enter].

Load samples in the respective rack position.

Repeat until all sample ID numbers are entered for the respective sample rack positions.

When all samples are programmed and loaded in the respective sample rack positions, press [Quit] on the numeric keypad.

With all reagents and consumables on board, place the sample rack into the right rack pool of the sampler (input tray).

Press [Start] or [Register More] (during operation) to begin processing.

Sample ID number (barcoded sample) read by barcode reader/
No automatic inquiry of tests (no host connection or the host is down)

From the Main Menu, press [Worklist].

Verify that Register Rack is displayed above the parameter keys. If not, press [Rack].

Verify that the next available rack is displayed (white background). Use the [Prev] or [Next] key to locate the next available rack.

Use the up and down arrow keys to select the desired sample rack position.

Press the blue test key [DDi]. Continue to select tests to be performed on the sample if necessary. Each test ordered should show the symbol "":

Press [Enter] and [Quit] to clear the numeric ID entry keypad.

Use the arrow keys to select samples for sample specific configurations:

To perform the sample in 'Micro Mode': For samples to be performed in the Micro mode, press the [Micro] key for each sample. A "" symbol will be displayed on the Micro key. Samples programmed to be performed in Micro Mode will display "M" in the "opt." column in the respective worklist position on the screen.

To perform a reflex test on a sample: Press the [Reflex] key. A "" symbol will be displayed on the Reflex key. Samples programmed for reflexive testing will display "R" in the "opt." column in the respective worklist positions on the screen.
Note: Reflexive testing cannot be performed on samples running in Micro Mode.

To remove a redilution or set a redilution, repeat analysis, and/or dilution ratio (MDA) for a particular sample: Press the [Sample Specific] key. From the 'Sample Specific' screen select the assay and select the desired re-analysis method or MDA setting for the sample. Press [Return] until the respective worklist is displayed.
Note: Redilution and dilution ratio options are only available on tests in which the test protocol includes a primary dilution.

Repeat process for all remaining sample rack positions, if necessary.

Load the barcoded sample in the respective sample rack position with the bar code facing the analyzer.

Press [Start] or [Register More] (during operation) to begin processing.

Sample ID number (barcoded sample) read by barcode reader /
Automatic Inquiry of tests (host connection is operational)

When the host computer is connected using bi-directional communication, host inquiry takes place when the sample ID is read and the analysis parameters are automatically registered.

Load the barcoded sample in the respective rack position with the barcode facing the analyzer.

Place the sample rack into the right rack pool of the sampler (input tray).

Press [Start] or [Register More] (during operation) to begin processing.

NOTE: The cause of any printed error code must be investigated and the appropriate corrective action taken prior to reporting results.

QUALITY CONTROL PROCESSING

Innovance D-Dimer Control 1 and 2 can be run from the sample rack or from a predefined reagent holder position.

From the Sample Rack

Innovance D-Dimer Control Plasma 1 and 2

From the Main Menu, press [Worklist].

Select the Rack Worklist by pressing the respective key: [Rack].

Use the up and down arrow keys to select the desired sample rack position.

Press [ID No. Entry].

Press the [QC] key on the numeric keypad and the quality control file number e.g. if the Level 1 control is configured for QC file number two, press [QC] and [06].

Press the [DDi] key.

Press [ENTER]. The Worklist will display the ID as a quality control file number, e.g. QC06.

Repeat steps above to program Innovance D-Dimer Control 2 (QC07).

If necessary, continue to program routine patient or other levels of quality control material.

Transfer the control material into a 4 mL analyzer cup. Place the control material in the corresponding position of the sample rack and load the rack.

Press [Start] or [Register More] (during operation) to begin processing.

From a Pre-defined Reagent Holder Position

The controls are usually run in the sample rack for D-Dimer. However, the following procedure can be used if the Innovance D-Dimer Control 1 and 2 are run from the reagent holder:

Load Control into the appropriate pre-defined reagent holder position:

Press the [Set Reagents] key from the 'Main Menu'. The 'Consumable' screen will appear.

Confirm that the lid signal (status of cover opening) is green; then open the light shield lid.

Verify that the appropriate group (Group 1 or 2) is displayed on the 'Consumable' screen. The appropriate group will have the QC file number, e.g. [QC06], displayed.
Note: If the appropriate group is not displayed, press [Select Group] to display the 'Group Change' screen.

'Group Change' screen: Press the correct group key, then [Return]. Verify the reagent position key displayed in pink on the 'Consumable' screen contains the correct reagent.

Press [QC06] reagent holder position key. The reagent holder position is displayed in the upper left corner of the reagent holder position key. The position is designated with a letter D and a position number.

Pour the control material into a 4 mL analyzer cup. Place the cup in the appropriate reagent holder position.

Verify that the correct lot number is displayed. To change the lot number, Press [Lot #], enter the correct information and press [Enter].

Verify that the correct expiration date is displayed. To change the date, press [Exp.Date], enter the correct data in the format mm/dd/yyyy, and press [Enter].

Verify that the correct vial type is displayed. To change it press [Vial], and use the arrow keys to select the correct type and press [OK].

Press [ENTER] on the numeric keypad. The reagent holder position key for the control will be displayed with a green background.

From the Main Menu, press [Work List] to display the 'Work List' screen.

Press [Reagent Holder] to display the 'Work Load List' and use the arrow keys to select QC06.

Press [DDi]. The symbol "" will be displayed, indicating that Innovance D-Dimer has been selected for QC analysis.

Press [Start] or [Register More] (during operation) to begin the analysis.

REPORTING RESULTS:

The D-Dimer concentration in mg/L is calculated automatically by the analyzer based on the reference curve. The D-Dimer level is expressed as initial fibrinogen equivalent unit (FEU). An FEU is the quantity of fibrinogen initially present that leads to the observed D-Dimer level. Increases in D-Dimer concentration observed with thromboembolic events can be variable due to localization, extension and age of the thrombus. Therefore, a thromboembolic event cannot be excluded with certainty solely on the basis of a D-Dimer concentration being within the reference range of ostensibly healthy persons.

Record and report patient and quality control values according to laboratory procedure.

Hemolyzed, lipemic, or icteric samples is noted with the result as a result message or specimen message.

Reference Range:

Reference Range values determined for D-Dimer are:

Less than or equal to 0.59mg/L FEU for non ED Patients

Less than 0.50 mg/L FEU for ED Patients

Cut-off value for D-Dimer is: 0.50mg/L FEU

PROCEDURE NOTES:

Overall performance of D-Dimer testing is dependent on reagent and instrument performance. Acceptable variability (imprecision) should be such, that the total coefficient of variation (CV) of the analytic system is less than 20% on the same lot of control plasma.

A new reference curve should be established with each change of reagent lot and change of instrument or with any deviation from control or proficiency testing limits.(Every 6 months). Recalibration occurs if there are any problems with the curve, or outlier points. One point can be repeated or a new set of reagents/calibrator made up and run. Troubleshoot with the hot line for further issues.

D-Dimer concentrations below 3.65 mg/L FEU obtained by redilution measurement have to be confirmed without redilution.

The Innovance D-Dimer total measuring range is defined by the vendor and the concentration of the calibrator used and is approximately 0.19 to 4.40 mg/L FEU. The measuring range can be extended to approximately 35.20 mg/L FEU by automatic redilution of samples above 4.40 mg/L FEU. Samples with concentrations above 35.20 mg/L FEU can be further diluted manually with Innovance D-Dimer Diluent. We report results out as a result of: >34.00mg/L FEU.

All components of the Innovance D-Dimer Kit are lot dependent. The combination of lots other than those specified may lead to incorrect results.

LIMITATIONS OF THE PROCEDURE:

Turbidity and particles in plasma may interfere with the determination. Therefore plasmas containing particles must be centrifuged for 10 minutes at approximately 15,000 x g again prior to testing.

Lipemic samples or samples containing particles that cannot be clarified by centrifuging must be excluded from testing.

Higher levels of lipids or turbid samples can lead to falsely elevated or decreased values.

Patient samples may contain heterophilic antibodies (e.g. human anti-mouse antibodies (HAMA) and rheumatoid factors) that could react with immunoassays to give a falsely elevated or depressed result. This assay has been designed to minimize interference from heterophilic antibodies. Nevertheless, complete elimination from this interference cannot be guaranteed

Results of the D-Dimer test should always be interpreted in conjunction with the patient’s medical history, clinical presentation, and other findings. Clinical diagnosis should not be based on he results of INNOVANCE D-Dimer alone.

Patients with a distal DVT may have a normal INNOVANCE D-Dimer result.

Interfering Substances

Levels of the following do not interfere with the Innovance D-Dimer assay:

Analyte	Interference Up to
Bilirubin	15 mg/dL
Hemoglobin	100 mg/dL
Triglycerides	600 mg/dL

General Reagent Specificity

There is no significant effect on the determination of D-Dimer due to levels of the following:

Analyte	Interference Up to
Creatinine	30 mg/dL
Heparin, sodium	3.0 U/mL
Rheumatoid Factors	1330 IU/mL
Albumin	6 g/L
Fibrinogen	1000 mg/dL
Urea	500 mg/dL
Uric Acid	20 mg/dL
Immunoglobulin G (IgG)	5 g/dL

REFERENCES

Innovance D-Dimer package insert. Dade Behring. Marburg, Germany, April 2007

Innovance D-Dimer Control package insert. Dade Behring. Marburg, Germany, April 2007.

Dade® CA System Buffer package insert. Dade Behring. Marburg, Germany, May 2008

Dade® Owrens Veronal Buffer package insert. Dade Behring. Marburg, Germany, May, 2008

Clinical Laboratory Standards Institute. Collection, Transport and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays: Approved Guideline-Fourth Edition.
CLSI Publication H21-A5. Wayne, PA, January, 2008

Sysmex® Operator’s Manual CA-1500 Automated Coagulation Analyzer. No. 461-2662-6. Kobe, Japan, October 2003

Application Sheet for Innovance D-Dimer on CA1500 Automated Coagulation Analyzer