For Research Use Only in the US and Canada Page 1 of 15 ML-08-00002 Rev 01

RUO T-TAS® plus

Overview The RUO T-TAS® plus system consists of a laptop (Windows 7) and a micro pump instrument with video camera and uses a single-channel cartridge chip. The system is only available as a 3 component system (computer/instrument/camera) and the PC contains the T-TAS plus proprietary software. Whole blood thrombus formation is measured using 2 different chips; an AR chip coated with collagen and tissue thromboplastin to measure thrombus formation as platelet aggregation and fibrin deposition and a PL chip coated with collagen to measure only platelet aggregation as the thrombus event. Thrombus formation is quantitated by the start time of occlusion, the total time to thrombus occlusion, and the total thrombus as AUC. The whole blood sample is pipetted into the sample reservoir and then an overcap is attached to capture excess whole blood. Part of the overcap containing the excess blood is detached and discarded and the sample reservoir is attached to the chip. Mineral oil in the pump line attached to the sample reservoir is used to push blood through the chip at a defined flow rate. After flow through the chip, the spent blood is collected in the chip (PL) or a waste tank (AR). The system software records the pressure (up to ~150 kPa) throughout the test and the CCD camera records the thrombus formation during intervals of the test. The chip is heated via a heating platen above and below the chip. The RUO T-TAS plus system checks pressure/flow with a Teflon block where mineral oil is pumped and specific check parameters must be met. The system software provides an output of the system check indicating nominal operation or an error. The CCD camera is 10X magnification. The foci of thrombus formation can be evaluated with an image analysis software, the ZIA program, which processes the video recording segments into still images and then patches the images into a single file of the thrombus formation along with image quantitation. Multiple thrombus formation tracings can be viewed together using the ResultViewer software.

For Research Use Only in the US and Canada Page 2 of 15 ML-08-00002 Rev 01

Output Parameters

1. T10…..time to 10 kPa above base pressure. This is the Occlusion Start Time. 2. OT…..total Occlusion Time is the time to reach base pressure plus 60 kPa for the PL

chip (T60) and the time to reach base pressure plus 80 kPa for the AR chip (T80). 3. AUCXX…..Area Under the Curve at xx minutes. For the PL chip, AUC10 is the

cumulative pressure recorded during the 10 minute assay. The AR chip records cumulative pressure for 30 minutes as AUC30. (Note: If the test reaches 60 kPa/80 kPa before the scheduled assay time of 10/30 minutes, then the AUC is the measured kPa plus the kPa at 60 kPa/80 kPa for the remaining duration of the scheduled assay time.)

4. kPa/min…..change in pressure between T10 and OT.

For Research Use Only in the US and Canada Page 3 of 15 ML-08-00002 Rev 01

AR Chip The AR chip has one channel and is coated with collagen (type I) and tissue thromboplastin and is 300 µm wide by 80 µm deep by 15 mm length. The assay requires 3.2% citrated whole blood (specific vacutainer collection tube provided). For the assay, 480 µL of citrated blood is mixed with 20 µL of Corn Trypsin Inhibitor/Ca (provided), and then 450 µL of the blood solution is immediately added to the sample reservoir and the test begun. The test requires an AR sample reservoir, AR overcap, and AR waste tank and is performed at 37° C for 30 minutes. Two flow rates are available for the AR chip:

Wall Shear Rate Flow Rate 600 sec-1 10 µL/min 240 sec-1 4 µL/min

The software (in automatic recording mode) shoots the entire flow path at 3 minute intervals for a total of 10 image segments. Manual image recording is also available. PL Chip Each PL chip has two channels for 2 separate assays, but only one test can be run at any time. Each assay (320 µL) requires hirudin whole blood (specific vacutainer collection tube provided), PL sample reservoir, and a PL overcap. The test is performed at 36° C for 10 minutes. The chip is coated with collagen (type I) and consists of 25 micro channels, each 40 µm wide by 40 µm deep, over a channel length of 1.5 mm. Three flow rates are available for the PL chip:

Wall Shear Rate Flow Rate 2000 sec-1 24 µL/min 1500 sec-1 18 µL/min 1000 sec-1 12 µL/min

The software shoots the entire flow path (in automatic recording mode) at 1 minute intervals for a total of 10 image segments. Manual image recording is also available. Additional Materials Mineral Oil is used for both assays and a 25 mM EDTA solution is only used with the AR chip. These materials are purchased from DiaPharma. Distilled water is used to flush the EDTA line when the instrument is not in use.

For Research Use Only in the US and Canada Page 4 of 15 ML-08-00002 Rev 01

Chips and T-TAS plus components

For Research Use Only in the US and Canada Page 5 of 15 ML-08-00002 Rev 01

PL Chip Configuration

PL chip…..

PL chip with heater raised…..

For Research Use Only in the US and Canada Page 6 of 15 ML-08-00002 Rev 01

Representative Data

AR Chip…..

For Research Use Only in the US and Canada Page 7 of 15 ML-08-00002 Rev 01

For Research Use Only in the US and Canada Page 8 of 15 ML-08-00002 Rev 01

Data Files for AR Chip….

T-TAS CONDITION & RESULT

CONDITIONStart Date 2/3/2015Start Time 14:00:24

Sample NameCommentProgram No 1 (number)Upper Heater Temperature

Set Point 37 (degree C)Start Value 36.9 (degree C)

Lower Heater TemperatureSet Point 37 (degree C)Start Value 37.1 (degree C)

lighting brightness 65 (%)

RESULTBase Pressure 2.1 (kPa)Occlusion Start Time 0:06:08 (h:m:s)Occlusion Time 0:07:40 (h:m:s)Occlusion Speed 45.7 (kPa/min)10.0(kPa) 0:06:08 (h:m:s) 6.13320.0(kPa) 0:06:24 (h:m:s) 6.40030.0(kPa) 0:06:39 (h:m:s) 6.65040.0(kPa) 0:06:52 (h:m:s) 6.86750.0(kPa) 0:07:04 (h:m:s) 7.06760.0(kPa) 0:07:17 (h:m:s) 7.28370.0(kPa) 0:07:28 (h:m:s) 7.46780.0(kPa) 0:07:40 (h:m:s) 7.667Limit Pressure 150.0(kPa) 0:00:00 (h:m:s) 0

Data Holder NameArea Under Thrombogram 1864.6/(min)

C:\T-TAS\Data\20150203\150203 140024 #37 AR(D80-40) 10ul No2Z(1145) CV Lot141203O-174\

#37 AR(D80-40) 10ul No2Z(1145) CV Lot141203O-174

T-TAS plus AR chipFile from: Data.150203 140024.#37 AR(D80-40) 10ul No2Z(1145) CV Lot141203O-174

MEASURED DATADate time measured upump 1 upump 2 CF pump1 CF pump2 stirrer process measure average UpperTemp LowerTemp

pressure flow rate flow rate flow rate flow rate revolution time enableNo. y/m/d h:m:s kPa uL/min uL/min % % % minute deg C deg C deg C

0 15/02/03 14:00:24 0 100 0 0 0 0 0.02 1 0 36.9 371 15/02/03 14:00:25 10.5 100 0 0 0 0 0.02 1 0 36.9 372 15/02/03 14:00:26 10.4 100 0 0 0 0 0.03 1 0 36.9 373 15/02/03 14:00:27 10.4 100 0 0 0 0 0.05 1 0 36.9 374 15/02/03 14:00:28 18.3 100 0 0 0 0 0.08 1 0 36.9 375 15/02/03 14:00:29 19 100 0 0 0 0 0.08 1 0 36.9 376 15/02/03 14:00:30 19.3 100 0 21 0 0 0.12 1 0 36.9 377 15/02/03 14:00:31 19 100 0 21 0 0 0.12 1 0 36.9 378 15/02/03 14:00:32 4.9 10 0 21 0 0 0.15 1 0 36.9 379 15/02/03 14:00:33 2.8 10 0 21 0 0 0.17 1 0 36.9 3710 15/02/03 14:00:34 2.8 10 0 21 0 0 0.17 1 0 36.9 3711 15/02/03 14:00:35 2.5 10 0 21 0 0 0.18 1 0 36.9 3712 15/02/03 14:00:36 2.2 10 0 21 0 0 0.2 1 0 36.9 3713 15/02/03 14:00:37 2.3 10 0 21 0 0 0.22 1 0 36.9 3714 15/02/03 14:00:38 2 10 0 21 0 0 0.23 1 0 36.9 3715 15/02/03 14:00:39 2 10 0 21 0 0 0.25 1 0 36.9 36.916 15/02/03 14:00:40 2.3 10 0 21 0 0 0.27 1 0 36.9 36.917 15/02/03 14:00:41 2.2 10 0 21 0 0 0.28 1 0 36.9 3718 15/02/03 14:00:42 2.2 10 0 21 0 0 0.3 1 0 36.9 3719 15/02/03 14:00:43 2.2 10 0 21 0 0 0.32 1 0 36.9 3720 15/02/03 14:00:44 2 10 0 21 0 0 0.33 1 0 36.9 3721 15/02/03 14:00:45 2.4 10 0 21 0 0 0.35 1 0 36.9 3722 15/02/03 14:00:46 1.9 10 0 21 0 0 0.37 1 0 36.8 3723 15/02/03 14:00:47 2 10 0 21 0 0 0.38 1 0 36.8 3724 15/02/03 14:00:48 2.2 10 0 21 0 0 0.4 1 0 36.8 3725 15/02/03 14:00:49 1.8 10 0 21 0 0 0.42 1 0 36.8 3726 15/02/03 14:00:50 2 10 0 21 0 0 0.43 1 0 36.8 36.927 15/02/03 14:00:51 2.2 10 0 21 0 0 0.45 1 0 36.8 36.928 15/02/03 14:00:52 1.9 10 0 21 0 0 0.47 1 0 36.8 3729 15/02/03 14:00:53 2 10 0 21 0 0 0.48 1 0 36.8 3730 15/02/03 14:00:54 2 10 0 21 0 0 0.5 1 0 36.8 37

For Research Use Only in the US and Canada Page 9 of 15 ML-08-00002 Rev 01

AR chip Image

AR chip, images at 1, 2, and 3 minutes.….

For Research Use Only in the US and Canada Page 10 of 15 ML-08-00002 Rev 01

Representative Data

PL Chip…..

For Research Use Only in the US and Canada Page 11 of 15 ML-08-00002 Rev 01

Data Files for PL Chip…..

T-TAS CONDITION & RESULT

CONDITIONStart Date 9/24/2015Start Time 14:10:54Sample Name T-TAS!18 PL 18ul Control-sampleCommentProgram No 13 (number)Upper Heater Temperature

Set Point 36 (degree C)Start Value 35.8 (degree C)

Lower Heater TemperatureSet Point 36 (degree C)Start Value 35.9 (degree C)

lighting brightness 15 (%)

RESULTBase Pressure 5.2 (kPa)Occlusion Start Time 0:01:59 (h:m:s)Occlusion Time 0:04:08 (h:m:s)Occlusion Speed 23.3 (kPa/min)10.0(kPa) 0:01:59 (h:m:s) 1.98320.0(kPa) 0:02:13 (h:m:s) 2.21730.0(kPa) 0:02:25 (h:m:s) 2.41740.0(kPa) 0:02:56 (h:m:s) 2.93350.0(kPa) 0:03:39 (h:m:s) 3.65060.0(kPa) 0:04:08 (h:m:s) 4.13370.0(kPa) 0:00:00 (h:m:s) 080.0(kPa) 0:00:00 (h:m:s) 0Limit Pressure 80.0(kPa) 0:00:00 (h:m:s) 0

Data Holder NameArea Under Thrombogram 438.3/(min)

C:\T-TAS\Data\20150924\150924 141054 T-TAS!18 PL 18ul Control-sample\

T-TAS plus PL ChipFile from: Data.150924 141054.T-TAS!18 PL 18ul Control-sample

MEASURED DATA

Date time measured upump 1 upump 2 CFpump1 CFpump2 stirrer process measure average UpperTemp LowerTemppressure flow rate flow rate flow rate flow rate revolution time enable

No. y/m/d h:m:s kPa uL/min uL/min % % % minute deg C deg C

0 15/09/24 14:10:54 0 100 0 0 0 0 0.03 1 0 36.2 361 15/09/24 14:10:55 10.5 100 0 0 0 0 0.03 1 0.5 36.2 362 15/09/24 14:10:56 10.6 100 0 0 0 0 0.03 1 1 36.2 363 15/09/24 14:10:57 11 100 0 0 0 0 0.05 1 1.6 36.2 364 15/09/24 14:10:58 11.7 100 0 0 0 0 0.07 1 2.1 36.2 365 15/09/24 14:10:59 17.9 100 0 0 0 0 0.08 1 3 36.2 366 15/09/24 14:11:00 21.9 100 0 0 0 0 0.1 1 4.1 36.2 367 15/09/24 14:11:01 21.6 100 0 0 0 0 0.13 1 5.2 36.1 368 15/09/24 14:11:02 21.4 100 0 0 0 0 0.13 1 6.3 36.1 369 15/09/24 14:11:03 22.2 100 0 0 0 0 0.15 1 7.4 36.1 3610 15/09/24 14:11:04 10.1 18 0 0 0 0 0.18 1 0.5 36.1 3611 15/09/24 14:11:05 7.2 18 0 0 0 0 0.18 1 0.8 36 3612 15/09/24 14:11:06 6 18 0 0 0 0 0.2 1 1.1 36 3613 15/09/24 14:11:07 6 18 0 0 0 0 0.22 1 1.4 36 3614 15/09/24 14:11:08 5.4 18 0 0 0 0 0.23 1 1.7 36 3615 15/09/24 14:11:09 5.4 18 0 0 0 0 0.25 1 2 36 3616 15/09/24 14:11:10 5.2 18 0 0 0 0 0.27 1 2.2 36 3617 15/09/24 14:11:11 5.3 18 0 0 0 0 0.28 1 2.5 36 3618 15/09/24 14:11:12 5.1 18 0 0 0 0 0.3 1 2.7 36.1 3619 15/09/24 14:11:13 5 18 0 0 0 0 0.32 1 3 36.1 3620 15/09/24 14:11:14 5.2 18 0 0 0 0 0.33 1 3.2 36.1 3621 15/09/24 14:11:15 5.2 18 0 0 0 0 0.35 1 3.5 36.1 3622 15/09/24 14:11:16 5 18 0 0 0 0 0.37 1 3.8 36.1 3623 15/09/24 14:11:17 5.3 18 0 0 0 0 0.38 1 4 36.1 3624 15/09/24 14:11:18 5.2 18 0 0 0 0 0.4 1 4.3 36 3625 15/09/24 14:11:19 5.2 18 0 0 0 0 0.42 1 4.5 36 3626 15/09/24 14:11:20 5.4 18 0 0 0 0 0.43 1 4.8 36 3627 15/09/24 14:11:21 5.1 18 0 0 0 0 0.45 1 5.1 36 3628 15/09/24 14:11:22 5.3 18 0 0 0 0 0.47 1 5.3 36 3629 15/09/24 14:11:23 5.2 18 0 0 0 0 0.48 1 5.6 36 3630 15/09/24 14:11:24 5.1 18 0 0 0 0 0.5 1 5.3 36 36

For Research Use Only in the US and Canada Page 12 of 15 ML-08-00002 Rev 01

PL chip Image

PL chip, time images at 1, 2, 4, 6, and 8 minutes.….

For Research Use Only in the US and Canada Page 13 of 15 ML-08-00002 Rev 01

Summary of AR and PL Assay Features

For Research Use Only in the US and Canada Page 14 of 15 ML-08-00002 Rev 01

Frequently Asked Questions What are the materials needed for a PL assay or AR assay? One PL chip package kit consists of 15 chips and there are 2 PL tests per chip. Each PL chip package requires PL sample reservoirs (30)/overcaps (30). The whole blood must be drawn in a hirudin vacuum collection tube (3 mL draw); these are packaged as 50 tubes. One AR chip package kit consists of 15 chips and there is one AR test per chip. Each AR chip package requires AR sample reservoirs (15)/overcaps (15) and waste tanks (15). The whole blood must be drawn in a specific 3.2 % citrate vacutainer tube (4.5 mL draw, Terumo, 100 tubes). The assay also requires CTI/Ca solution (0.6 mL) and a solution of 25 mM Na EDTA which the user prepares. Note that the sample reservoir differs for the PL and the AR chip. And that there are specific blood collection tubes to use for each test! How are reagents shipped and stored? Reagents (chips and CTI/Ca solution) are shipped overnight on frozen ice packs. Upon receipt, the chips and CTI/Ca solution are stored at -20° C or colder. The sample reservoirs/overcaps/waste tanks and the hirudin vacutainer tubes and the 3.2% citrate vacutainer tubes are stored at room temperature. EDTA chemical/EDTA solution and mineral oil are stored also at room temperature. Why are there different anticoagulants to use with each chip? The PL test is designed to measure primary hemostasis. The coagulation protein cascade is inhibited by adding hirudin (a direct thrombin inhibitor) to the whole blood. Platelets are activated by binding to the collagen-coated chip and the activated platelets aggregate as a white cellular clot. The AR test is designed to measure cumulatively primary and secondary hemostasis. Platelets aggregate as the AR test is a collagen-coated chip and it is also coated with tissue thromboplastin. As the whole blood is collected in 3.2% citrate, calcium must be added at the start of the assay to allow secondary hemostasis to proceed via initiation by thromboplastin of the extrinsic pathway. Corn Trypsin Inhibitor is added to the whole blood to inhibit Factor XII and the intrinsic pathway. Why are there different wall shear rates for the PL and AR chip? The shear rates are designed to approximate the blood shear rates in-vivo. For the PL chip, platelet aggregation occurs in the arteries and blood shear rates change depending on the size of the artery. In comparison, secondary hemostasis occurs primarily in the venous circulation at much lower shear rates and, again depending on the size of the vein, the in-vivo blood shear rates differ.

For Research Use Only in the US and Canada Page 15 of 15 ML-08-00002 Rev 01

How long is a sample viable for testing? A sample is tested 1 hour after draw to 3 hours after draw. The manufacturer is currently evaluating sample testing times less than 1 hour after draw. What are the reagent preparations before testing? As the PL and the AR chip is stored at -20° C or colder, they should equilibrate at RT for 30 minutes before use. Heating the chip on the instrument preheater can accelerate the RT equilibration. What are the maintenance requirements? On a daily basis when using the AR chip, the EDTA line/pump is purged with distilled water and stored in distilled water to prevent EDTA salt deposition. On an annual basis, the instrument tubing and filter on the EDTA line/pump is replaced.