Cleveland Family Study

3.6.4 Aliquoting Sample Tubes

Standard procedures as outlined below are followed for most Vacutainer tubes with anticoagulants such as EDTA, Citrate, and Silicon-coating for serum collection. However, the LCBR offers other collection tubes for hemostasis assays that have specific handling requirements:

• Aliquoting consists of removing the serum or plasma in small amounts (e.g.: 1.0 mL) by pipette and placing it into the appropriate color-coded cryovials (as outlined in cryovial specifications and the aliquoting schemes). Color-coding is predetermined and used to identify sample type such as EDTA plasma vs. Serum, etc.
• This process must be done while the tubes and cryovials are on ice (unless otherwise noted).
• When aliquoting serum and plasma from the centrifuged collection tube, be careful not to disturb the top of the cell layer with the pipette tip, as this will result in platelet, white cell and red cell contamination
• Use a new pipette tip for each draw tube.
• Once the sample is aliquoted, cryovials should be immediately frozen in an upright position at -70°C or promptly placed on dry ice for quick freezing.
• If any tubes are accidentally mixed during pipetting so that the plasma is contaminated with red cells, they may be re-centrifuged.

3.6.4.1 Procedures for Special Anticoagulant Tubes (SCAT-I, SCAT-II)

• Tube must be refrigerated until use.
• This tube must be drawn after at least one other tube (such as the serum tube) has been drawn, or draw a small tube (3 mL) and discard prior to the special tube.
• This tube is ‘non-sterile’. It must be drawn using a butterfly apparatus with 12 inches of tubing; alternatively, a syringe may be used for the venipuncture, and expressed through the cap (with great care to limit turbulence) into the SCAT tube.
• Mix the tubes for 30 seconds ASAP after blood draw; keep on ice until processing.
• Follow standard processing procedures to obtain plasma.

3.6.4.2 Procedures for Platelet Tube (Diatube H)

• Tubes must be stored in a refrigerator or put on ice for at least 15 minutes before blood collection. This tube is also collected using a butterfly apparatus with 12 inches of tubing.
• After centrifugation (30,000 g-minutes), the plasma from this tube must be filtered.
• Use a 3 cc syringe with Luer adapter (no needle) and remove the plunger.
• Attach a filter (0.2 micron Gelman Acrodisk) to the end of the syringe.
• Using a calibrated, plastic transfer pipet, draw up approximately 1.0 mL from the middle region of the plasma in the Diatube and place it into the bottom of the syringe.

• Insert the syringe plunger and slowly push the plasma through the filter into a cryovial. It will become steadily harder to push the plunger through. Avoid pushing through the foam at the end.
• Dispose of the syringe and filter in biohazardous waste container.
• Freeze the filtered plasma in an upright position immediately at –70°C.

3.6.4.3 Any Citrate Tube for Factor VIIc Assays

• Factor VIIc has been shown to be affected by incubation at 4ºC (i.e., on ice or in the refrigerator). Consequently, if the most accurate assay is desired, care should be taken to use room temperature handling and processing.

• Samples should not be snap-frozen in a dry ice bath or in a –70°C freezer, once preparation is complete. When room temperature handling and processing is not available, a standard processing at 4°C may be used with an effort to limit the time the sample is in the cold, with the understanding that some degree of activation will occur.

3.6.4.4 Any Citrate Tube for PAI-1 Assays

• PAI-1 is present in platelets, so that care must be taken to avoid platelet contamination. Specifically at least 30,000 g-min of centrifugation should be used.
• Plasma should be withdrawn from the tube carefully, avoid the last ¼ ” before the cell pellet, as this is where the platelets are located.
• Care should be taken to avoid shaking the tube in any way, to avoid re-mixing the platelets with the plasma layer.