Sample Collection and Deproteinization – 2,3-DPG
Reagents
Perchloric acid, 0.6M, refrigerated
Potassium carbonate, 2.5 M
Supplies
2 centrifuge tubes, 10 ml, label A, B
cryovial, 2ml
2 ml Heparin Vacutainer (green top)
Ice bucket with ice
Pipettes, 5ml, 1ml
Shipping materials
Centrifuge set at 4oC
1. Before drawing the blood sample, Pipette 5 ml ice-cooled 0.6M Perchloric
acid Into a 10 ml centrifuge tube, label A, place on ice with empty vacutainer.
2. Collect blood from vein into the ice-cooled vacutainer tube until it is full. Mix
well, place back on ice.
3. Immediately remove 1 ml of blood to the acid in tube A and mix well.
Note: Flush pipette by repeated filling and emptying.
4. Centrifuge the mixture in tube A at 5,000 g for 10 minutes at 4 oC.
5. Remove 4 ml of the clear, colorless supernatant to a new tube (B) and
neutralize by adding 0.5 ml 2.5 M Potassium carbonate.
6. Place the new tube (B) in the ice and keep cold for at least 60 minutes.
7. Centrifuge the mixture in tube B at 5,000 g for 10 minutes at 4 oC to remove
the perchlorate precipitate.
8. Transfer 1 ml of the new supernatant to a cryovial and store refrigerated until
assayed or shipped. Clearly identify each sample.
9. Note: 2,3-DPG is stable for at least 1 day in the neutralized extracts, so
samples need to be sent the same day by courier or by overnight FedEx to
the laboratory.
10. Fill out the requisition form completely.
11. Insert the cryovial/s into a biohazard ziplock bag containing an absorbent
material and seal the top of the bag.
12. Place the bag inside the foam cooler with two frozen gel packs.
13. Close the foam cooler and place the original requisition form on top of the
foam cooler. A copy of the form should be kept at the site.
14. Tape the outer cardboard box closed.
15. Apply all appropriate shipping labels to the outside of box.
16. If shipping, complete the FedEx air bill and schedule a FedEx pickup:
http://www.fedex.com At the same time, send an email to
RBClab@CHORI.org to inform the lab to expect a sample.
Frans Kuypers RBC Lab Room 1114
Children's Hospital Oakland Research Institute
5700 Martin Luther King Jr. Way
Oakland, CA 94609
Phone: 510-450-7621
Perchloric Acid, 70% from Sigma-Aldrich catalog #244252 at $50.3
for 50 ml. Dilute 1.16x to 60% or 0.6M by adding 8.3 ml of distilled
water.
Potassium Carbonate from Sigma-Aldrich catalog # 367877 at
$41.20 for 10g. Dissolve 3.45g in 10 ml distilled water to make a
2.5 M solution. Store refrigerated, will be enough for 20 samples.