TITLE: Blood Draw
AUTHORS: Clemens Scherzer, Bradley Hyman, Charles Jennings

[DESCRIPTION]
This protocol explains the Standard Operating Protocol for Blood Drawing.

[GUIDELINES]
PROTOCOLS NO LONGER IN USE 

SERUM 
MATERIALS: One 4 ml yellow top SST Gel and Clot Activator SERUM tube (BD Franklin Lakes, NJ., Ref# 367977) PROTOCOL: After centrifugation, pipette 500 uL of the liquid phase above the formed clot into each of the four tubes labeled “SE-01, SE-02, etc.” Avoid contaminating the sample by preventing the pipette tip from touching the wax that the serum sits upon. Tipping of the tube can ease the access to the remaining serum volume that sits on top of the wax layer. The remainder of the tube can be discarded. 


FREEZER STORAGE
  
Freezers are divided into 4 shelves, with 6 racks per shelf, and 24 boxes that can be held in each shelf. In total, 576 boxes, approximately 2,160 sample sets, can be stored in one -80°C freezer. The first three shelves are designated by visit number: Shelves A1-6 (top shelf) house samples from enrollment visits, shelves B1-6 (2nd shelf) house samples from the 1st year follow-up, and shelves C1-6 (3rd shelf) house samples from the 2nd year follow-up. Shelves D1-6 contain packed red blood cell tubes (PRBC), DNA, and RNA, extracted from blood as described in the protocols above. CSF is designated between two freezers in selected racks. Freezer storage and transactions of samples are recorded in the Freezerworks Inventory software.

[STEPS]
SECTION: Blood Draw
1. Store all tubes at Room temperature (18ºC to 25ºC) before use.

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2. Label all tubes with the subject ID# prior to the blood draw.
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3. The PAXgene™ Blood RNA Tubes should be the first tubes drawn in the phlebotomy procedure. Collect all other tubes after collecting the PAXgene™ Blood RNA Tubes in the following order: 10 ml EDTA, one 8.5 ml black and blue 8.5 ml CPT tube, two 6 ml EDTA, 2 ml EDTA, 2.7 ml EDTA and 2.7 ml blue top (only if interested in doing an LP). In special cases, one 8.5 ml Solution A will be substituted for one 6ml purple top EDTA-BD Vacutainer tube (only taken once per subject if applicable). When applicable, a 2.7 ml EDTA tube is taken last.
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4. Prior to blood draw, ask donor three questions: 
Are you allergic to latex? 
Do you take blood thinners? 
Do you have a history of fainting during blood draws? 
If donor answers yes for any question, take appropriate precautions. Ask donor to sit, place pillow (covered with blue pad) beneath arm. Examine donor’s arm for optimal puncture site, then apply tourniquet. Using gloves, clean site with alcohol wipes.
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5. Using a blood collection set and a holder, collect blood into the first PAXgene™ Blood RNA Tube using your institution’s recommended procedure for standard venipuncture technique. 

The following techniques shall be used to prevent possible backflow:
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5.1. Place donor’s arm in straight, downward position.
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5.2. Hold tube in a vertical position, below the donor’s arm during blood collection.
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5.3. Release tourniquet as soon as blood starts to flow into tube.
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5.4. Make sure tube additives do not touch stopper or end of the needle during venipuncture.
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6. Allow at least 10 s for a complete blood draw to take place in each tube. Ensure that the blood has stopped flowing into the tube before removing the tube from the holder.

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7. Immediately after blood collection, gently invert/mix (180 degree turns) the tubes 8 – 10 times.
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8. REPEAT STEPS 4 to 6 for each of the tubes to be collected per subject.  . 

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9. Remove needle in quick motion and dispose in Sharps Disposal Container. Immediately press gauze to puncture site, ask donor to apply pressure and raise arm (keeping it straight) until blood flow stops. Bandage with Latex-Free Bandaids or gauze and tape.
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10. Record time/date of draw on specimen collection form. Attempt to keep all tubes upright.
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11. Place the 8.5 ml Solution A tube in a separate biohazard bag along with the requisition form including gender, date drawn, and date sent information. Ideally within 4 days of the draw, it should be delivered by cooler to the lab that forms the cell line. It should be kept at Room temperature (18ºC to 25ºC) until then.

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12. The 2 ml purple top CBC will be sent in a separate biohazard bag the same day of the draw to the core lab for processing immediately along with the proper requisition forms and labeling. Reticulocytes are requested at the enrollment visit only.
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13. The rest of the tubes are placed in a biohazard bag (double bag if necessary) and are brought to the lab by cooler within 3 hours of the draw for processing and storage.
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14. 2.7 ml blue top tube is placed on ice in a specimen cup and sent to the core lab for immediate PTT/PT/INR processing with the proper requisition forms and labeling.