Whole Blood, Plasma, and Buffy Coat Processing

For PAXgenes:

1. Label the collection tubes with the sample ID as appropriate.

2. Place aliquot tubes on dry ice prior to procedure so they are pre-cooled.

Before centrifuging the 10ml BD purple top tube , pipette 550µL aliquots of whole blood into two Eppendorf tubes labeled “WB-01 and WB-02.”

Place the 10 ml BD purple top and the two 6 ml BD purple tops into the blue racks and, using the Eppendorf centrifuge 5810R set to 2000rcf,25°C.

10 ml BD purple top tube. After centrifugation, pipette 500µL into Eppendorf tubes labeled “PL-01, PL-02, etc” (8-10 tubes depending on the sample). Avoid introducing bubbles into the tube, as it will make it harder to pipette the plasma. Stop taking plasma when the buffy coat begins to enter the pipette tip. If the amount of plasma in the tip is less than 500 uL, place the remaining amount into the last plasma tube labeled “PL-16.” Switch to a 100 uL pipette and remove any excess plasma that is sitting above the buffy coat. Place this excess plasma in the last plasma tube labeled ”PL-16.”

With the 100 uL pipette, pipette approximately 200-250 uL of buffy coat into one Eppendorf tube labeled “BC-01.” (Try to avoid pipetting up the pellet as much as possible; however, some pellet will be unavoidably pipetted up with the buffy coat.) If more than 250 uL of buffy coat can be extracted, place the remaining volume of buffy coat into the other buffy coat tube labeled “BC-02.”

1st 6 ml purple top tube. ^st 6 ml purple top tube. After centrifugation, pipette 500µL into the remaining, empty plasma tubes. Avoid introducing bubbles into the tube, as it will make it harder to pipette the plasma. Stop taking plasma when the buffy coat begins to enter the pipette tip. Switch to a 100 uL pipette and remove any excess plasma that is sitting above the buffy coat. Any excess plasma can be equally distributed amongst the 16 plasma tubes.

With a 100 uL pipette, pipette approximately 200-250 uL of buffy coat into the tube labeled “BC-02.”

2nd 6 ml purple top tube. ^nd 6 ml purple top tube. After centrifugation, pipette 500µL into the remaining empty plasma tubes. Avoid introducing bubbles into the tube, as it will make it harder to pipette the plasma. Stop taking plasma when the buffy coat begins to enter the pipette tip. Switch to a 100 uL pipette and remove any excess plasma that is sitting above the buffy coat. Any excess plasma can be equally distributed amongst the 16 plasma tubes

With a 100 uL pipette, pipette approximately 200-250 uL of buffy coat into the tube labeled “BC-03.”

Split each set of tubes into two boxes and label according to their H number. The various aliquots will be placed into each box and scanned as follows:

1. Box 1 (labeled with a * and stays here): Whole Blood 01, Plasma 01-08, and Buffy Coat 01-03
2. Box 2 (goes to backup freezer): Whole Blood 02, and Plasma 09-16
3. PRBC (placed in designated box in -80C) The number of aliquots per type of aliquot is dependent upon the sample volume. If an uneven number of aliquots per type of aliquot are produced, then more of the aliquots will be placed in Box 1. (ex: 9 plasma aliquots total = 5 plasma in Box 1 and 4 plasma in Box 2).

Place aliquots into the -80°C within 4 hours of the blood draw.