Preparing plasma from a whole blood sample begins with centrifugation, a technique that separates components based on density. Plasma is the pale yellow, liquid portion of blood, representing over half its total volume. To prevent clotting, blood is collected in tubes containing an anticoagulant. Centrifugation spins the sample at high speeds, forcing heavier cellular components—red blood cells, white blood cells, and platelets—to the bottom, leaving the less dense plasma at the top for collection.
Understanding Relative Centrifugal Force
The effectiveness of separation is determined by the force applied to the sample, not just the rotor speed. Centrifuges use two primary measurements: Revolutions Per Minute (RPM) and Relative Centrifugal Force (RCF), measured in units of gravity (‘g’). RPM is inconsistent because it only indicates the number of rotations per minute; different sized machines spinning at the same RPM apply vastly different forces. RCF is a standardized measurement accounting for both RPM and the centrifuge rotor’s radius. This calculation determines the actual gravitational force exerted, ensuring that a protocol specifying 1,500 x g achieves the same results regardless of the centrifuge model used.
Standard Timing and Speed Protocols
The time required to centrifuge a blood sample depends directly on the RCF applied, as a stronger force achieves separation faster. For routine clinical plasma preparation, a widely accepted protocol involves spinning the sample for 10 to 15 minutes at 1,000 to 2,000 x g. This range is sufficient to create a clean, distinct barrier between the cellular pellet and the plasma layer. Separation must occur quickly; most clinical assays require plasma to be separated from cells within one to two hours of the blood draw to prevent cellular components from altering the sample.
Platelet-Poor Plasma (PPP) Preparation
Specific analytical requirements often demand more aggressive centrifugation parameters to produce platelet-poor plasma (PPP), which is necessary for coagulation testing. To ensure a minimal number of platelets remain suspended, protocols call for a higher RCF or a longer duration. A common PPP standard uses 1,500 x g for 15 minutes. Some laboratories may use a force as high as 3,000 x g for 10 to 20 minutes to achieve more complete platelet depletion.
Factors That Modify Centrifugation Time
Standard protocols must often be adjusted based on the downstream test or collection method. The type of anticoagulant used, such as EDTA, Heparin, or Citrate, influences sample processing. Tubes containing a separation gel barrier also modify the protocol, requiring a specific RCF to move the gel into position. This forms a stable seal between the plasma and the cell pellet.
Temperature control is another factor that modifies the procedure, as some analytes are heat-sensitive and can degrade if the sample warms during spinning. While some centrifugations occur at room temperature, samples for molecular biology or sensitive protein analysis are often spun in refrigerated centrifuges maintained at 4°C.
Handling Plasma After Separation
Once centrifugation is complete, immediate handling of the plasma is necessary to maintain sample quality. The separated plasma (supernatant) must be carefully removed, or aliquoted, into new, clean tubes. When drawing off the plasma, avoid disturbing the buffy coat, the thin, whitish layer just above the red blood cells. This layer contains white blood cells and platelets, and their contamination can skew test results.
The plasma is generally transferred using a pipette, taking care not to touch the cellular layer to ensure it remains cell-free. After aliquoting, the plasma must be handled according to its intended use and storage needs. For immediate analysis, the plasma is often kept refrigerated at 2 to 8°C. For long-term storage, aliquots should be frozen rapidly at -20°C or colder, and repeated freeze-thaw cycles must be avoided to preserve sample integrity.