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SUPPLEMENT:

Systems Biology Coagulopathy of Trauma and Burn Investigators’ Manual of Operations

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General Table of Contents 1-Overview and Background ............................................................................................................... 3

1A-General Overview ....................................................................................................................... 3 1A1-General Safety Issues and Precautions for Handling Blood Specimens .............................. 3 1A2-General Organization ........................................................................................................... 3

1B-Materials ..................................................................................................................................... 4 1B1-Equipment ............................................................................................................................ 4 1B2-Supplies ................................................................................................................................ 4 1B3-Reagents for TEG/ROTEM ................................................................................................... 5

2-Blood Collection 2A-Tube Labeling ............................................................................................................................. 5 2B-Phlebotomy Logistics .................................................................................................................. 5 2C-Phlebotomy Details ..................................................................................................................... 5 2D-Phlebotomy Master Schedule ..................................................................................................... 6 2E-Phlebotomy Master Schedule for Thermal Injuries ..................................................................... 6

3- Processing & Local Storage of Biomarker Blood Samples ............................................................. 6 3A-Overview of Processing .............................................................................................................. 6 3B-Transferring the Tubes From Collection Site to Processing ....................................................... 8 3C-Centrifugation for Plasma and Cell Sample Preparation ............................................................ 8 3D-Aliquoting .................................................................................................................................... 8 3E-Nucleic Acid Sample Preparation ............................................................................................... 9

3E1-RNA Preservation from PAXgene or Tempus tubes ............................................................ 9 3E2-DNA Preservation from Cell Pellets or PAXgene tubes ....................................................... 9

3F Local Sample Storage ............................................................................................................... 10

4-TEG/ROTEM .................................................................................................................................. 11 4A-Assay and Instrument Principle ................................................................................................ 11

4A1-Instrument Principle ROTEM ............................................................................................. 11 4A2-Instrument Principle TEG .................................................................................................. 11

4B-Assay Principle for Systems Biology Protocol .......................................................................... 11 4C-Reagent Preparation ................................................................................................................. 12 4D-Instrument Set Up ..................................................................................................................... 13

4D1-Assay Start ......................................................................................................................... 13 4D2-TEG Experiments ............................................................................................................... 13 4D3-ROTEM Experiments ......................................................................................................... 15

5-Shipping of Blood Samples to Collaborating Laboratories ............................................................. 17 5A- Shipping Protocol ..................................................................................................................... 17

6-Clinical Data Collection .................................................................................................................. 18

7-IATA Packaging and Shipping Guidelines ..................................................................................... 18

Page3of161. Overview and Background The “Systems Biology of Coagulation and Trauma-Induced Coagulopathy” project seeks to develop a comprehensive understanding of the fundamental, highly networked, molecular and physiological processes underlying normal coagulation and the perturbations of those processes that result in coagulopathy.

1A. General Overview

1A1. General Safety Issues and Precautions for Handling Blood Specimens

Laboratory safety protocols and procedures were followed In accordance with the Occupational Safety and Health Administration (OSHA) regulations on blood borne pathogens.

1A2. General Organization

All procedures performed are to be under IRB approved protocols and follow HIPPA regulations.

General design of blood collection and processing is illustrated in the schematic below:

1B. Materials

1B1. Equipment

1. Centrifuge with swinging bucket rotor (capable of achieving 5,000g) 2. Freezer (-80oC) 3. Refrigerator (4oC) 4. TEG/ROTEM Instrument (s)

a. TEG, with associated computer, or ROTEM machines to achieve 6 channels total (see *Note) b. ROTEM and TEG cups and pins (distributed as cup/pin units from supplier) c. Tips for the ROTEM automatic pipette c. Timer capable of counting up d. A set of calibrated pipettes (to include the range of pipetting volumes 2uL-1,000uL) and appropriately sized disposable pipette tips e. Water bath, 1L or larger, for thawing quench solution f. Forceps for removing the cup and pin from the quench solution

5. Tube racks, tube mixer, biohazard disposal bags for accumulated waste 6. Balance for weighing tubes (American Weigh Scales, AC-100 Digital Pocket Scale 100x0.01g, cat #AC-100)

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*Note: The details in this manual refer to the assumption of one ROTEM (ROTEM delta) machine and one TEG (Haemonetics TEG-5000) machine being available; however, any combination of the machines may be used. The ROTEM consists of four channels and is capable of running four independent conditions; the Haemonetics TEG provides two channels and is also capable of running independent conditions. To obtain six channels, either 2x ROTEM, 1x ROTEM and 1x TEG, or 3x TEG will be required. The Haemonetics TEG assay and ROTEM assay can be used interchangeably, or side-by-side, for the protocols in this manual. If TEG machines are used where ROTEM is specified in this manual, all volumes must be adjusted to account for the greater total cup volume (the TEG holds 360uL while the ROTEM holds 340uL). The activator volumes must be adjusted to maintain the concentrations present in the ROTEM, ex: 21.18uL of ExTEM and InTEM must be added to TEG channels to account for the greater volume. 1B2. Supplies

1. Evacuated blood collection tubes 5mL SCAT (Haematologic Technologies cat. # SCAT-144-4.5/5)** 2.7mL Citrate (BD cat. # 363083) 2.5mL PAXgene (BD cat. # 762165) and/or 2.5mL Tempus (BD cat # 761165) 2. Syringes for drawing off blood from the lines: 20 mL (Fisher Scientific cat # 22-257-127) 3. 1.5 mL cryovials (Fisher Scientific cat # 02-681-338) and caps (cat # 02-681-358 and 02-681-359) 5. Study labels containing unique label numbers 6. Freezer boxes 7. Pipette tips, tube racks, gloves, disinfectant wipes

*Each site should keep a detailed log of batch tube weights for each lot number of evacuated blood collection tubes. Upon receipt of a new lot of tubes, 10 tubes from that lot should be weighed and their weights recorded.

**Note: The SCAT tubes are not sterile and under no circumstance may be connected to a patient’s line! These tubes must be filled by syringe.

1B3. Reagents for TEG/ROTEM 1. Calcium chloride solution for recalcification (CaCl2) for both ROTEM and TEG* 2. Hepes Buffered Saline solution (HBS) for a) diluting tissue factor (Tf) for both ROTEM and TEG 3. Low-concentration in-house tissue factor (Tf) * 4. High-concentration Tf reagent (ExTEM) manufactured by ROTEM 5. Calcium reagent (StarTEM) manufactured by ROTEM

2. Blood Collection

2A. Tube Labeling

Each blood draw corresponds to a unique “draw number” (e.g. draw number 0001 will be the first blood draw). In addition, each aliquot that is produced from that draw has a unique number, and a

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unique barcode or label. Aliquots are filled following a standard sequence noted both on a paper case report form (CRF).

2B. Phlebotomy Logistics

Phlebotomy will be performed by EMS personnel in the pre-hospital setting, and clinical RN or trained research staff in the other locations (ED or ICU). Ambulances are stocked with evacuated tubes and syringes in kits.

Research technicians are present for all draws after the patient’s arrival to the hospital. The technicians will provide syringes to the nursing staff at the appropriate times and record draw information in the CRF, including:

1. Information on patient location (EMS, ED, or ICU) 2. Information on line used for blood collection (peripheral IV, central line, or arterial line) 3. Information on time variables (time from tube filling to centrifugation, aliquotting, and freezing) 4. Information on the quality of each tube sample collected

2C. Phlebotomy Details

Note: Attention should be paid to minimizing turbulence whenever possible. Small steps such as slanting the needle in the vacutainer to have the blood run down the side of the tube instead of shooting all the way to the bottom, may result in significant improvement in results. 1. The first draw off a patient’s line at a given time point, should discard the first mL (for

venipuncture) or 3 mL (for central line) using a 5 mL syringe. If other blood draws on the patient were already performed this step is not necessary.

2. Draw > 22 mL blood into a 20 mL syringe (the syringe has the capacity to overfill by a couple of mL).

3. Rapidly use the syringe to fill evacuated collection tubes. 4. If a direct venipuncture has been used instead of a central line, we recommend use of a butterfly

assembly. Secure the needle in the arm and proceed to fill the tubes sequentially. For SCAT tubes (not sterile), attach a syringe (needle removed) to the butterfly line to fill the syringe then subsequently fill the SCAT tube.

5. All citrate and SCAT tubes are inverted 3-4 times then placed in a tube rack at room temperature (25°C) for transport to the laboratory. The PAXgene DNA tube must be inverted 8-10 times and similarly placed in a rack at room temperature for transport to the laboratory.

6. Immediately upon arrival to laboratory these tubes should be placed on a balance and their weights recorded on the CRF.

2D. Phlebotomy Master Schedule

The phlebotomy schedule lists time points for blood sample collection. Pretreatment blood, either from EMS or upon arrival to the ED, will be considered time 0 for the purposes of this study. It is essential to have pretreatment blood samples for inclusion in the study; all other samples will be obtained as feasible (e.g. patient care, disposition, or death may preclude complete sampling).

Sample 1 2 3 4 5 6 7 8 9 10 11

Time 0 2hr 4hr 6hr 8hr 12hr 24hr 48hr 72hr 96hr 120hr

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Location ED/ OR/ ICU

ED/ OR/ ICU

OR/ ICU

ICU ICU ICU ICU ICU ICU ICU ICU

2E. Phlebotomy Schedule for Thermal Injuries

Burn patients only will be followed for additional time-points after 24hrs, collected every 12hrs, for 7 days, and then day 14 and day 21 after admission.

Sample 7B 8 8B 9 9B 10 10B 11 11B 12 Etc.

Time 36hr 48hr 60hr 72hr 84hr 96hr 108h 120h 132h 148h “”

Location ICU ICU ICU ICU ICU ICU ICU ICU ICU ICU “”

3. Processing and Local Storage of Biomarker Blood Samples

3A. Overview of Processing

Each time point will use an identical set of evacuated tubes, labeled with a unique ID number. Tubes will be filled and processed immediately according to the following priority list:

Tube No.

Type Purpose Filled Volume

Cap Color

Processing Final Aliquots

1 SCAT Plasma proteins (*and cellular nucleic acid where possible; see 3C and 3E below)

~4.5 mL Clear Spin to separate PPP and cell pellets

0.5 mL

2 Citrate #1 “ “ 2.7 mL Blue “ “ 0.5 mL

3 Citrate #2 “ “ 2.7 mL Blue “ “ 0.5 mL

4 Citrate #3 “ “ 2.7 mL Blue “ “ 0.5 mL

5 Citrate #4 “ “ 2.7 mL Blue “ “ 0.5 mL

6 Citrate #5 On-site TEG/ROTEM 2.7 mL Blue ROTEM and/or TEG n/a

7 PAXgene RNA or Tempus RNA

mRNA prep 2.5 mL Red Freeze immediately n/a

8 PAXgene DNA*

DNA prep (*when cell pellet separation as

2.5 mL Red Freeze immediately n/a

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described in 3C and 3E is not possible)

Each tube will be scored using the following quality control system:

Three parameters routinely assessed:

1. Adequate filling of SCAT/citrate tubes (determined by weight) 2. Processing time 3. Hemolysis

For each parameter, a score of “1” or “0” will be assigned. Criteria for each parameter is as follows:

1. Weight must be within the following ranges: a. SCAT tubes: 10.2-11.3 grams (within 5% of the target weight of 10.8 grams) b. Citrate tubes: 9.7-10.7 grams (within 5% of the target weight of 10.2 grams) c. PAX tubes: 17.1-19.6 grams (within 5% of the target weight of 18.6 grams)

2. Processing time must be 15 minutes or less a. Processing time defined as the time between the draw and centrifugation or incubation

3. Hemolysis is not present a. Only tubes that are centrifuged will be assigned a score for this parameter.

For each parameter that falls within the guidelines stated above, a score of “1” is assigned. Therefore, the “ideal” sample that fits all criteria listed above will receive a “3” (or a “2” if the tube is not centrifuged.) Each score will be noted on the CRF.

Number of good parameters Total score

All 3 parameters good (only for centrifuged tubes) 3

2 parameters good 2

1 parameter good 1

0 parameters good 0

3B. Transferring the Tubes from Collection Site to Processing

Transportation of blood samples to the laboratory for further processing and on-site TEG/ROTEM should be accomplished within 10 minutes. No more than 15 minutes should elapse between blood draw and processing. The tubes are to be transported at room temperature.

3C. Centrifugation for Plasma and Cell Sample Preparation (Citrate #1, #2, #3, #4, and SCAT)

Note: The purposes of centrifugation are: 1) to separate plasma from cells, including most of the platelets; 2) provide cellular material for preparation of DNA.

There are two options for centrifuging the tubes, choice A or choice B:

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A) If possible, the SCAT tube should be subjected to a ‘soft’ spin, the plasma removed, and then the plasma subjected to a further ‘hard’ spin along with four of the citrate tubes. The cell pellet obtained from the SCAT tube ‘soft’ spin will undergo the Nucleic Acid Sample Preparation protocol in section 3E2 option A.

The procedure is as follows:

1. Centrifuge SCAT tube at room temp (25°C) at 400g for 10 min with brake OFF. Transfer plasma to a clean tube and:

2. Centrifuge citrate tubes #1, #2, #3, and #4 and the SCAT plasma from step 1 above (SCAT tube) at room temp (25°C), for 50,000 g-min (3,000g for 17 minutes)

3. Once centrifugation is complete, carefully remove tubes from centrifuge and place in tube rack.

4. Aliquot the plasma as described under section 3D and treat the SCAT tube cell pellet as described under section 3E2 option A. Freeze citrate tube cell pellets as described in section 3E2 option A.

B) If the on-site Nucleic Acid Preparation is not feasible, perform only the ‘hard’ spin on all

five tubes: 1. Centrifuge citrate tubes #1, #2, #3, and #4 and the SCAT tube at room temp (25°C), for

50,000 g-min (3,000g for 17 minutes) 2. Once centrifugation is complete, carefully remove tubes from centrifuge and place in tube

rack. 3. Aliquot the plasma as described under section 3D and treat the pellet as described under

section 3E2 option B.

3D. Aliquotting Note: Proper labeling is critical on the cryovials into which the separated plasma is pipetted. Labels must be guaranteed to withstand freezing and thawing. Most standard office printers are not capable of printing in a permanent ink. Writing directly on the cryovials (i.e., not using a label) is not recommended. Aliquotting consists of removing the plasma in 0.5mL increments by pipette and placing it into the appropriate cryovials with color-coded O-ring caps for optimal seal.

1. Plasma from the SCAT and citrate tubes can be aliquotted directly from the draw tube. 2. This process is to be done with the tubes and cryovials at room temperature 3. Be careful not to disturb the top of the cell pellet with the pipette tip, as this will result in platelet,

white cell and red cell contamination; try to avoid ~3 mm of sample from the very top of the cell layer as this contains the bulk of the platelets.

4. Use a new pipette tip for each draw tube type (citrate; SCAT). 5. Once the sample is aliquotted, cryovials should be frozen in an upright position immediately at -

80°C. 6. There are separate instructions for continuing to process and store the separated cell pellets in

section 3E.

3E. Nucleic Acid Sample Preparation (PAXgene tubes, Citrate #1, #2, #3, #4, and SCAT)

3E1. RNA Preservation from PAXgene RNA or Tempus Tubes

PAXgene RNA or Tempus tubes are used for RNA preservation. Upon draw the tube is inverted 8-10 times. The tube should immediately be placed into the -80°C freezer (no further handling).

3E2. DNA Preservation

Page9of16If cell separations as described in 3C (option A) and 3E (below) are not possible at the site, and an additional tube can be accommodated based on total volume of draw:

PAXgene DNA tubes are used for DNA preservation and immediately upon draw the tube is inverted 8-10 times. The tube can be placed into the -80°C freezer (no further handling).

Alternatively:

After processing the plasma from the citrate and SCAT tubes as described in section 3C+D, the cell pellets that are remaining in the tubes are processed for DNA preservation. There are two options for processing these pellets:

Option A. For SCAT tube (whose pellet was only subject to a “soft” spin) perform red blood cell lysis prior to freezing, per protocol below. NOTE: This protocol is adapted from Dr. Brownstein, which required a blood draw involving a 7 mL EDTA tube. All reagents should be appropriately scaled down to reflect the actual draw volume. Reagents - Lysis Buffer (EL Buffer), Qiagen Cat No 79217; provided in 1 Liter containers. Store at room temperature. Do not use if solution has any color. - Sterile transfer pipette, polyethylene, Fisher cat No 13-711-20 - “RLT Plus” Buffer, Qiagen; Cat No 1053393 - 12 ml syringe,Monoject, Cat No 512878 - 18 g, 1 1/2 inch needle, Becton-Dickinson

Methods

1. Using a polyethylene transfer pipette, transfer the plasma, buffy coat, and packed cells (approximately 4 mL total) immediately to each of two 50 mL conical tubes (Corning Labs. Cat No 430828) (2 mL each) and add 45 mL of room-temperature Lysis Buffer to each of the conical tubes. (Qiagen EL Buffer). 2. Mix the samples thoroughly by vortexing the tightly capped tube and place it on ice for 15 minutes, vortexing the tube gently at five and ten minutes during that period. 3. Centrifuge the samples in a swinging bucket, refrigerated centrifuge at 4oC for 10 minutes at 400g, with the brake OFF. 4. Decant the supernatant carefully, removing all of the fluid, and discard into a biohazard waste container, taking care not to disturb the cell pellet. Add 15 mL of room-temperature Lysis Buffer to each of the two tubes, and vortex gently to resuspend the pellets. 5. Centrifuge the samples in a swinging bucket, refrigerated centrifuge at 4oC for ten minutes at 400g, with the brake OFF. 6. Decant the supernatant carefully, removing all of the fluid, and discard into a biohazard waste container, taking care not to disturb the cell pellet. Loosen cell pellet by gently tapping the side of the tube on a hard surface, then flick the bottom of the tube with your forefinger while holding the tube near the top with the fingers of the other hand. 7. Add 2 mL of room-temperature Buffer RLT Plus to each tube (Qiagen, RNeasy® Midi Kit Cat No 75142). Vortex each tube briefly to ensure cell lysis. Using a 5 mL pipette, combine the contents of each tube into one tube; final volume approximately 4 mL.

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8. Homogenize the pellet in the Buffer RLT Plus at room temperature by aspirating and forcing the sample repeatedly through an 18 g needle (Becton-Dickinson cat No 305199) (minimum ten times). The needle should be on a 10 or 12 mL syringe (Monoject, cat No 512878). Try to keep the foaming/bubbles to a minimum. 9. Place a barcode label on the tube and immediately store at -80°C until ready to ship to the Collaboration Site.

Option B. For citrate tubes #1, #2, #3, and #4, and SCAT freeze cell pellets immediately at -80 oC in

the original draw tubes.

3F. Local Sample Storage All frozen samples should be stored frozen at -80°C until aliquots are processed or shipped to collaborating sites. Samples are preserved using screw-top storage tubes with barcodes and labels which are stable at -80°C.

Page11of164. TEG/ROTEM (Citrate #5)

4A. Assay and Instrument Principle

Measurements of the viscoelastic properties of clot formation and clot lysis in whole blood samples are obtained using either rotational thromboelastometry (ROTEM machine) or thromboelastography (Haemonetics TEG machine). This manual illustrates both ROTEM and TEG methods but any combination of machines may be used; when conducted under identical conditions by skilled technicians, and the output of these machines is equivalent.

4B. Assay Principle for Systems Biology Protocol

Blood will be drawn by hospital staff via either venipuncture or through a pre-established line at indicated time points. Blood will be immediately citrated to prevent clotting prior to assay time. When the assay is performed, the blood that has been collected into the citrate tube must now be recalcified to overcome the citrate, thus providing calcium essential to clotting, and in most cases will contain an activator to initiate clotting.

Each draw will consist of six experiments; they are summarized in the table below and described in detail in this section:

4D. Instrument Set Up

The ROTEM and TEG machines must be on for at least 15 min prior to assay time to allow the cuvette holders to reach 37°C. For the Systems Biology protocol, they should remain on at all times.

4D1. Assay Start

This protocol starts with the acquisition of blood drawn into a 2.7mL evacuated sodium citrate tube, as outlined in section 3A, yielding a final sodium citrate concentration of 10.88 mM. Complete a CRF for each assay.

4D2. TEG Experiments Before each TEG assay, verify that the machine is level by inspecting the bubble-level on the top of the machine (see Figure below). Also verify that both channels are at 37°C (temperature monitor if found on front of machine).

Figure: The diagram on the right illustrates a TEG machine that is not level

1. ROTEM- High-concentration tissue factor stimulated blood (ExTEM)

2. ROTEM- High-concentration tissue factor stimulated blood (ExTEM)

3. ROTEM- Low-concentration tissue factor stimulated blood (In-House)

4. ROTEM- Low-concentration tissue factor stimulated blood (In-House)

5. TEG- High-concentration tissue factor stimulated blood (ExTEM)

6. TEG- Low-concentration tissue factor stimulated blood (In-House)

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The software should be programmed as follows:

1. Label the TEG channels: a. First line: ACIT_number_initial (example: ACIT001AA)1 b. Second line: hour of draw_reagent type (examples: EMSTF or 0EXTEM)

2. Vortex CaCl2 for TEG and Tf dilution vial for TEG 3. Check the TEG lever to make sure it is in the LOAD position 4. Secure cups in holder

a. Place cup in white plastic holder b. Seat the cup with both your thumbs c. Place your right hand on top of the machine,

and with left hand slide the plastic holder to the top of the channel

d. Press the button on the bottom of the plastic holder

e. Then bring the white plastic holder down and push down on either side of the plastic holder to seat the cup in the channel

5. Make sure channel 1 is selected for TEG Software a. Pipette 21 μL of StarTEM reagent into the first TEG cup b. Change pipette tip c. Pipette 21 μL of ExTEM reagent into the first TEG cup d. Change pipette tip e. Pipet 318 μL of blood into the first TEG cup f. Complete the following steps in QUICK succession (no more than 10 seconds):

i. Place pipette tip on the bottom of the cup and slowly aspirate and eject to mix the contents of the cup, avoid bubbles

ii. Set the pipette on counter iii. Load the cup (don’t click the button again)2 iv. Move lever to test position v. Click F10 on TEG screen vi. Eject the pipette tip to biohazard trash

6. Make sure channel 2 is selected for TEG Software a. Pipette 20 μL of TEG CaCl2 reagent (small eppendorf tube) into the second TEG cup b. Change pipette tip c. Pipette 20 μL of TEG Tf reagent into the second TEG cup d. Change pipette tip

1Ifthisisthefirsttimepatient’sbloodisbeingrun,theprogramwillaskyouifyouwanttocreatethenewpatient–click“yes”andinputpatient’sACITnumberinpop-upwindowagain,thenclick“Done”2Clickingthebuttonseatsthesmallerpartofthecuponthepin.Clickingitagainejectsthesmallerpartofthepin.

Figure illustrating the three lever positions on the TEG machine; the left channel is prepared for loading cups

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e. Pipet 320 μL of blood into the second TEG cup f. Place pipette tip on the bottom of the cup and slowly aspirate and eject to mix the

contents of the cup g. Complete the following steps in QUICK succession (no more than 10 seconds):

i. Place pipette tip on the bottom of the cup and slowly aspirate and eject to mix the contents of the cup, avoid bubbles

ii. Set the pipette on counter iii. Load the cup (don’t click the button again), iv. Move lever to test position v. Click F10 on TEG screen vi. Eject the pipette tip into the biohazard trash

7. Press “Done” in upper right corner to go to TEG curve screen 8. Run the TEG for 1.5 hours 9. Press “Stop” for each channel and then “TEG” icon on TEG software to save data 10. Remove cups and pins for holders and discard in biohazard trash 11. Clean the workspace and machines

IT IS IMPERATIVE THAT THE CHANNEL IS STARTED IN THE SOFTWARE IMMEDIATELY AFTER THE CARRIER IS SLID UP AND THE LEVER MOVED TO TEST POSITION! THIS SHOULD BE PRIORITIZED BEFORE DISCARDING THE PIPETTE TIP.

Once all samples have been started, press “done” on the TEG screen to view curves and parameters as they are established.

Run the channels for 1.5 hours. Stop channels after 1.5 hours by pressing “stop” on the TEG screen for each channel. Eject the cups and pins by moving the lever to “load” and pressing downward (see previous figure). Slide carrier down and press firmly past the first stop, ejecting the cup and pin; discard of cups and pins in biohazard waste.

4D3. ROTEM Experiments

1. The ROTEM instruments must be in “measurement” mode; the compatibility with automatic pipette use must be confirmed (can be adjusted in “setup” module)

Channel Labeling and Computer Settings

2. Label the ROTEM channels

a. Patient ID: ACIT_number_initial (example: ACIT001AA) b. Sample: hour of draw_reagent type (examples: EMSTF or 0EXTEM)

Initiator ‘Test type’ to set in software

ExTEM reagent (high-conc. Tf) ExTEM

ExTEM reagent (high-conc. Tf) ExTEM

In-house Tf reagent ExTEM

In-house Tf reagent ExTEM

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c. Comments: Technician name and unanticipated events 3. An adequate supply of all disposables (tips, cups/pins, biohazard waste bags) should be within

reach 4. Load cups immediately prior to obtaining blood (this allows for sufficient time before assay for

automatic calibration, while minimizing possibility of dust falling into cup): take whole cup unit from storage drawer, gently and firmly drive it into the pin, set the cup in the holder and seat firmly with blue tool

5. TIGHTLY secure cups in ROTEM holders using blue plastic piece (push straight down hard) 6. Vortex CaCl2 for ROTEM and Tf dilution vial for ROTEM 7. Fill ROTEM cups using the automatic pipet (See detailed instructions below)

a. Channel 1: StarTEM, ExTEM, blood b. Channel 2: StarTEM, ExTEM, blood c. Channel 3: ROTEM CaCl2, ROTEM Tf, blood d. Channel 4: ROTEM CaCl2, ROTEM Tf, blood

*When drawing up any fluid, be sure the tip is sufficiently deep into the fluid for the entire volume to be drawn up. If air is drawn up (any bubbles present) the entire sample must be restarted. Watch the pipette tip during aspiration.

*When expelling fluid, be sure the tip is NOT in the fluid or at the level which it will fill to. After ejecting fluid, the pipette fires once more to ensure that all fluid is out of tip- if the tip is in the expelled fluid, it will draw some back up and the required volume will not have been ejected.

i. Start the pipetting sequence by selecting the right-hand channel and pressing “Start. ii. Press the pipette firmly into a clean tip. iii. Place the tip well into the appropriate reagent and press the central blue button on

the pipette, then press the button for air cushion; next place the pipette tip into the activator and press the blue button one more time.

iv. Place the tip over the cup so that the fluid will be expelled into the bottom center of

the cup. Do not touch the bottom of the cup. Press the blue button. v. Eject tip (either side-button on the pipette) and replace with a new tip. vi. Invert citrate tube of blood end-over-end three times and place pipette tip well into the

blood (note: it is important that the tip is at least ½ way into the vial; 300uL will be pulled out, and the appropriate volume of blood will not be drawn if the pipette draws up any air). Press blue button.

§ No need to recap and invert blood between channels

vii. Rest the pipette tip on the rim of the ROTEM cup so that the blood runs down the

side of the cup when expelled. Do not place the tip far into the cup. Press the blue button.

viii. Without replacing the pipette tip, place the tip at the bottom edge of the cup and

press the blue button to draw up all the fluid in the cup. ix. Expel blood back into cup as before.

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x. Using hand not holding the pipette, slide incubation chamber (containing the cup) up until it magnetically attaches to the machine unit. Press okay in the dialog box that has popped up (note: you have less than 30 seconds to slide the chamber up and hit okay before the entire run is terminated).

xi. Eject tip into biohazard waste container and use the touch-screen to move to the

next channel.

xii. Repeat steps i-xi for each channel, using the appropriate reagents for that experimental channel

xiii. Immediately clean up any drops or spills.

Run the channels for a total of 1.5 hours. Stop the channels after 1.5 hours by pressing “stop channel” for each channel (machines can be programmed to terminate after 1.5 hours automatically). Save the data by pressing “clear channel” for each channel. Lower the cup holders by firmly pressing the blue clip on the chamber (this will “grab” the pin) and lowering the unit. Pop the cup/pin out of the chamber by pressing it against the ejection post, and discard cups and pins in biohazard waste container.

Note: There may not be sufficient volume for all 6 channels, as care should be taken not to exceed the approved amount of blood for the draw (5 mL); the two TEG channels should be run first, followed by the ROTEM channels in the order they are described. If blood volume is not sufficient, the uninitiated sample should be omitted.

8. Run the ROTEM for 1.5 hours 9. Press “clear channel” for each of the ROTEM channels to save data 10. Remove cups and pins for holders and discard in biohazard trash

5. Shipping of Blood Samples to Collaborating Laboratories

5A. Shipping Protocol

These instructions pertain to frozen blood samples shipments between collaborating sites. Plan to ship samples on Monday or Tuesday via Fed Ex Priority overnight or Approved Courier.

1. Line shipping container with absorbent material (i.e. lab mat, or paper toweling) 2. Place approximately ~5 to 10 lbs of dry ice on the bottom of the shipping container. 3. Place another layer of absorbent material (i.e. lab mat) on top of the dry ice – so it will be

between the dry ice and the freezer boxes. 4. Collect the freezer boxes containing samples to be shipped, and check the sample ID

numbers against the Shipping Form for that shipment. 5. Wrap absorbent material around the box and secure with a rubber band around the box. 6. Place each freezer box in a ziplock plastic bag and seal tightly. 7. Place ziplocked freezer boxes in the shipping container. Note: the ziplock bags should NOT be

in direct contact with the dry ice. 8. Add another layer of absorbent material on top of the freezer boxes in the shipping container. 9. Add remaining dry ice to the shipping container. Close and tape the Styrofoam lid. 10. Seal Phlebotomy/Processing Forms in a ziplock bag and place on top of the Styrofoam lid.

Include a cover sheet with recipient address and contact information. 11. Close the top of the outer cardboard sleeve of the shipping container with packing tape. 12. Affix shipping labels (Fed Ex label, Biological Specimen Category B UN3373 label, Dry Ice Class

9 UN1845 label, and Keep Frozen label) to outside of shipping container.

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13. Add extra shipping tape over the labels to ensure they will not fall off in transit. 14. Fill out the Shipping Log including the Fedex airbill #s and fax or scan and email to the receiving

site on the day samples are shipped. NOTE: This shipping protocol follows the procedures mandated by the International Air Transport

Association’s (IATA’s) Dangerous Goods Regulations-Packaging Instructions 650 and 904. 6. Clinical Data Collection

After enrolment of the patient is confirmed by obtaining an informed consent, clinical data is being recorded using REDCap Software, version 4.14.4, by research associates.

7. IATA Packing and Shipping Guidelines

http://www.iata.org/whatwedo/cargo/dangerous_goods/Pages/infectious_substances.asp