Phlebotomy · Processing · 6 min read · Open lesson
Serum, Plasma and Clotting Before Centrifugation
- Differentiate between serum and plasma in terms of composition, coagulation factors, and the collection tubes used to obtain them.
- Describe the pre-centrifugation clotting requirements for serum specimens, including required clotting times, proper tube positioning, and the effects of temperature.
- Identify post-centrifugation specimen layers, recognize visual sample interferences, and apply correct specimen handling procedures within the phlebotomist's scope of practice.
The Composition of Blood: Plasma versus Serum
In the human body, circulating blood consists of cellular elements suspended in a liquid matrix. When blood is drawn as whole blood, it contains plasma, red blood cells, white blood cells, and platelets. In clinical specimen processing, one of the most critical responsibilities of the phlebotomy technician is understanding whether an ordered test requires whole blood, plasma, or serum. Although plasma and serum may appear visually similar as clear or light-yellow fluids, they are chemically distinct due to the presence or absence of specific clotting proteins.
Plasma represents the liquid portion of unclotted, anticoagulated blood. To harvest plasma, blood is collected in a tube that contains an anticoagulant additive to prevent the coagulation process from taking place, followed by centrifugation. Because coagulation is intentionally blocked, plasma retains all of its natural clotting factors. Most notably, plasma contains fibrinogen, a soluble plasma protein, along with other essential coagulation factors such as prothrombin.
Serum, on the other hand, is the liquid portion of blood that remains after a specimen has been allowed to clot completely. Serum is obtained by drawing blood into a collection tube that does not contain an anticoagulant, allowing the natural coagulation process to finish, and then centrifuging the tube. A key difference between the two fluids is that plasma contains fibrinogen, while serum has little or no functional fibrinogen. During coagulation, the soluble protein fibrinogen is converted into an insoluble fibrin mesh to form the clot. Because fibrinogen is consumed during the formation of this blood clot, it is absent from the resulting liquid serum.
An everyday way to understand this difference is to think of a net catching items in a stream. In plasma, the materials to build the net are floating freely and dissolved in the fluid. In serum, the net has already been fully built and pulled out of the liquid, binding the cellular elements together into a solid structure and leaving the remaining fluid behind without its original dissolved building blocks.
| Feature | Plasma | Serum |
|---|---|---|
| Anticoagulant Used | Yes (preserves blood in an unclotted state) | No (allows blood to clot naturally) |
| Functional Fibrinogen | Present (along with other factors like prothrombin) | Little or no functional fibrinogen (consumed in the clot) |
| Pre-Centrifugation Clotting | No clotting period needed; can spin after mixing | Requires full clotting period before spinning |
| Separated Cellular Portion | Forms red blood cells and a distinct buffy coat | Compacts into an integrated cellular clot |
Collection Tubes and Centrifugation Layering
Specimen collection begins with verifying the patient's identity using at least two independent identifiers before performing venipuncture. Phlebotomists must also confirm that collection tubes are not expired prior to drawing blood. The choice of tube directly determines whether the processed sample will yield serum, plasma, or whole blood. Glass red-top tubes without additives yield serum after clotting and centrifugation. Serum separator tubes also yield serum, but they contain a clot activator and a thixotropic gel barrier to facilitate separation.
When plasma is required, tubes containing specific anticoagulants must be selected. Light-blue-top tubes contain the anticoagulant sodium citrate and yield plasma upon centrifugation. Green-top tubes contain the anticoagulant heparin and also yield plasma. Lavender-top tubes contain the anticoagulant EDTA and can yield plasma or provide whole blood. However, phlebotomists must note that whole blood specimens collected for a complete blood count must not be centrifuged, as the laboratory analyzer requires intact cellular elements evenly distributed in the fluid.
Immediately after collection, proper tube mixing is essential. Phlebotomists must perform prompt, gentle inversion of anticoagulant tubes to mix the additive with the blood, which reduces the risk of microclots. Inverting serum tubes with clot activators is equally important to ensure adequate distribution of the activator throughout the blood. Gentle inversion of collection tubes is strictly required to avoid mechanical hemolysis caused by vigorous shaking. The phlebotomist must also document the time of collection and processing according to facility protocol.
Centrifugation physically separates blood components based on density. Centrifuging an anticoagulated blood specimen separates it into three distinct layers: plasma on top, a buffy coat in the middle, and red blood cells at the bottom. Red blood cells settle at the very bottom of the centrifuged tube because they have the highest density. The buffy coat is the thin, whitish layer that forms between the plasma and red blood cells; it contains white blood cells and platelets and represents less than one percent of the total blood volume in a normal specimen. In contrast, centrifuging a clotted blood specimen separates it into serum and a compacted cellular clot, without a visible buffy coat.
Proper Clotting Procedures for Serum Specimens
Achieving high-quality serum requires strict adherence to pre-centrifugation protocols. Tubes intended for serum analysis must be allowed to clot completely before centrifugation. Furthermore, serum tubes must be allowed to clot in an upright, vertical position. Keeping serum tubes upright during clotting helps prevent the clot from adhering to the stopper, which maintains specimen integrity and facilitates clean separation during centrifugation.
The time needed for complete clotting depends on the tube type and its additives. Non-additive glass serum tubes generally require about 60 minutes to clot at room temperature, though technicians must always follow the tube manufacturer's instructions. Plastic serum separator tubes with silica clot activators typically require thirty minutes to clot completely. Specialized thrombin-based clot activator tubes can achieve complete clotting within five minutes.
Temperature plays a crucial role in the coagulation process. Phlebotomists must allow serum specimens to clot at room temperature rather than on ice. Chilling a serum tube during the clotting phase inhibits or delays the coagulation cascade, preventing the chemical reactions needed to produce a solid clot.
Patient health and medications can also influence clotting speed. Blood from patients taking anticoagulants may take longer to clot. Similarly, blood from patients with liver disease or severe coagulation factor deficiencies may clot more slowly than usual. Before centrifugation, check serum tubes for adequate clotting according to the tube manufacturer's instructions and laboratory protocol. If a serum specimen has not clotted completely, allow additional clotting time at room temperature according to laboratory protocol, and report a specimen that does not clot as expected to the laboratory supervisor.
Centrifugation Timing, Precautions, and Specimen Inspection
Timing is a key difference between handling serum and plasma tubes. Unlike serum tubes, plasma tubes do not require a clotting period and may be centrifuged after proper mixing, according to the test's processing instructions. This immediate centrifugation of plasma tubes makes them well suited for emergency or stat testing, where rapid laboratory results are critical for patient care.
Rushing the processing of serum tubes presents serious technical risks. Centrifuging a serum tube before clotting is complete can allow fibrin strands to form later in the separated serum. These latent fibrin strands in serum can obstruct sampling probes in automated laboratory analyzers, causing equipment downtime and testing delays. To prevent this, phlebotomists must never centrifuge incompletely clotted serum tubes. Additionally, do not manipulate a clot with an applicator stick; report incomplete clotting or separation according to facility protocol. Technicians must also remember not to re-centrifuge a gel-separator serum tube unless laboratory protocol permits it, because re-centrifugation can disrupt the barrier and contaminate the serum with cells.
After centrifugation is complete, the phlebotomist must inspect centrifuged serum or plasma for hemolysis, icterus, or lipemia. Hemolysis gives serum or plasma a pink or red appearance caused by the breakdown of erythrocytes. Lipemia gives serum or plasma a cloudy or milky appearance caused by high concentrations of lipids. Icterus can give serum or plasma a deep yellow to orange-brown appearance due to elevated bilirubin.
When processing or visual abnormalities are identified, the phlebotomist must act within their established role. The phlebotomist does not diagnose patient conditions or interpret laboratory results. Instead, the phlebotomist must document the time of collection and processing according to facility protocol and report specimen processing problems or abnormal specimen appearances to the laboratory supervisor.
Common mistakes
- Centrifuging a serum tube before the recommended clotting time has elapsed, which allows latent fibrin strands to form and obstruct automated analyzer probes.
- Placing a serum collection tube on ice to clot, which inhibits or delays the coagulation cascade instead of allowing it to clot at room temperature.
- Re-centrifuging a gel-separator tube after initial separation, which can disrupt the thixotropic gel barrier and contaminate the serum with cellular components.
- Shaking collection tubes vigorously instead of performing gentle inversions, causing mechanical hemolysis and erythrocyte breakdown that turns serum or plasma pink or red.
Summary
- Whole blood contains cellular elements (red blood cells, white blood cells, platelets) suspended in liquid plasma.
- Plasma is obtained from anticoagulated tubes (such as light-blue sodium citrate, green heparin, or lavender EDTA) and contains fibrinogen and prothrombin; it does not require a clotting delay before centrifugation.
- Serum is obtained from non-anticoagulant tubes (such as glass red-top or gel-barrier SSTs) and lacks functional fibrinogen because it is consumed during clot formation.
- Centrifuged anticoagulated blood forms three layers: top plasma, middle buffy coat (white blood cells and platelets, representing under one percent of volume), and bottom red blood cells.
- Serum tubes must clot vertically at room temperature (about 60 minutes for non-additive glass, 30 minutes for silica SSTs, or 5 minutes for thrombin tubes); chilling delays coagulation.
- Phlebotomists must never manipulate clots with applicator sticks, must not re-centrifuge gel tubes unless permitted by protocol, and must inspect specimens for hemolysis, lipemia, or icterus.
- All processing problems, incomplete clotting, and visual interferences must be reported to the laboratory supervisor while documenting collection and processing times according to protocol.
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