Attach 15. Study Visit 1 Blood Requisition Form

ATTACHMENT 15 STUDY VISIT 1 BLOOD REQUISITION FORM

MOM-le Study

VISIT 1 BLOOD SPECIMEN

(14.0 - 20 6/7 weeks gestation)

CRU Protocol #1331

Dr. Ann Borders Pager: (312) 695-1453

Name: __________________________

Study ID Number: ______________________________

CRU Maternal Log Number: ______________________________

Date: __________________________

C onfirm that participant has not used steroids (not including inhalers) in last 2 weeks:

C onfirm that participant has not experienced an illness, infection, or cold symptoms in last 2 weeks:

Assay	Tube	Comments from Phlebotomist	Research Assistant Directions	Aliquots	Comments from Lab Personnel
CRH	4 mL Purple-Top EDTA + Aprotinin		1. Add Aprotinin to blood tube. 2. Transport tube to processing lab within 30 minutes of collection.	1.5 mL in 2 mL cryovial
				remainder in 2 mL cryovial
Cortisol	10 mL Purple-Top EDTA		1. Transport tube to processing lab within 30 minutes of collection.	0.5 mL in 2 mL cryovial
CRP/EBV				0.5 mL in 2 mL cryovial
				0.5 mL in 2 mL cryovial
Cytokines				8 * 0.1 mL (strip tube)
				0.750 mL (cryovial)
DNA	8.5 mL Paxgene		1. Transport tube to processing lab within 30 minutes of collection.	no aliquots

Date/Time of Blood Draw: ______________________________

Date/Time Specimen Received in Lab: ______________________________

Specimen Received on Ice? YES / NO

Specimen Processed By: ______________________________

Cortisol, CRP/EBV and Cytokines (10mL EDTA Purple-Top Tube)

• Collect blood and gently invert tube 10 times to adequately mix blood with EDTA anticoagulant.

Processing Sample:

1. Adjust centrifuge to 3000rpm @ 4⁰C and spin for 15 minutes.

Aliquots (On Ice):

1. Pipette 0.5mL of plasma into 3 of the 2mL cryovials.

2. Pipette 0.75mL of plasma into the fourth 2mL cryovial.

3. Pipette 8 X 0.1mL of plasma into the strip tube.

4. Store additional samples in extra 2mL cryovials (store locally).

5. Immediately store all vials in -80ᵒC freezer.

6. Store strip tubes in a small box with 96 x 0.2mL racks with lids.

7. Use extra fine alcohol resistant marker to label the side of each tube (tubes can break off the strip when frozen).

8. Place a sturdy rubber band around the lid and box.

DNA (8.5mL Paxgene Tube)

• Collect blood and gently invert tube 10 times.

• No processing or aliquots necessary.

Storage of Aliquots:

1. Store Paxgene tubes using the styrofoam inserts and box in which they came.

2. Place at ~-20ᵒC for 24 hours and then store tubes in -80ᵒC freezer.

3. IMPORTANT: Place the box on its side when placed inside the freezer. Tubes may crack if upright.

CRH (4mL EDTA Purple-Top Tube + Aprotinin)

• Aprotinin aliquots will be prepared in advance. (Aprotinin from SIGMA A1153-100mg    $462.00)

• Make Aprotinin on ice and aliquot on ice. Dilute in the original bottle with 5mls of sterile saline. Mix well. Aliquot 35uL into each small 0.6mL tube for storage at -20ᵒC. Make sure to deliver liquid to bottom tube.

• This gives you a concentration of 20mg/mL or 1mg/50ul or 6TIU/50ul. (We need 1TIU/ml blood).

• These aliquots can be stored for up to 1 year.

On day of draw:

1. R emove Aprotinin from freezer, put on ice, and take to clinic.

2. If aliquot is still frozen when needed, gently roll tube in gloved hands to defrost.

3. C

   Plasma

   
   

   Buffy Coat

   
   

   Red Cells

   ollect blood and gently invert tube 10x to mix blood with EDTA anticoagulant.

4. Pipette 33uL of Aprotinin protease inhibitor into tube.

5. R ecap tube and gently invert tube 4x to mix blood with inhibitor.

6. Chill on ice until centrifugation.

Processing Sample:

1. S ample must be processed within 30 minutes of collection (if needed, sample can sit on ice for up to 1 hour).

2. Centrifuge at 3000rpm for 15 minutes at 4ᵒC.

3. Carefully remove tube from centrifuge as not to disturb buffy coat and note specimen color (yellow, pink or red).

4. Pipette 1.5mL of plasma (carefully so as not to disturb buffy coat) into the first 2mL cryovial and then pipette remainder into the second 2mL cryovial.

5. Immediately store all plasma cryovials in -80ᵒC freezer (or -20C if necessary).