How Is Serum Obtained? The Step-by-Step Process

Serum is obtained by drawing blood into a tube without anticoagulants, letting it clot, and then spinning it in a centrifuge to separate the liquid portion from the solid clot. The resulting clear, straw-colored fluid contains proteins, electrolytes, hormones, and other dissolved substances but lacks the clotting factors and blood cells found in whole blood. While that three-step summary sounds simple, each stage involves specific conditions that determine whether the serum is actually usable for laboratory testing, research, or therapeutic purposes.

What Makes Serum Different from Plasma

Blood drawn from a vein can yield two different liquid products depending on how it is handled. Serum comes from blood that has been allowed to clot naturally, so the clotting proteins (especially fibrinogen and its derivatives) get consumed during clot formation and are removed along with the blood cells. Plasma, on the other hand, is collected by adding an anticoagulant to the blood immediately, which prevents clotting altogether. Plasma therefore retains fibrinogen and other coagulation factors that serum does not.¹ This distinction matters because the choice between serum and plasma can affect the results of laboratory tests. Some analytes are more stable in one matrix than the other, and some assays are specifically validated for serum only.

Before the Needle: Pre-Collection Factors That Affect Results

The process of obtaining quality serum begins before any blood is drawn. Several patient-related variables can shift the concentration of substances in the blood enough to change test results, so standardizing these conditions is part of the process.

Body posture is one such variable. Moving from lying down to sitting or standing causes fluid to shift out of blood vessels and into surrounding tissues, which concentrates the blood. A study examining this effect found that switching from a supine to a standing position caused clinically meaningful increases in hemoglobin, hematocrit, red blood cell counts, and several types of white blood cells.² For most routine blood draws, sitting upright for several minutes before collection is the practical standard. But for tests where precision matters, labs may ask you to sit quietly for 10 to 15 minutes first.

Fasting is another consideration. Eating raises blood lipids and glucose, which can interfere with certain assays. Prolonged fasting and mild physical activity have been shown to cause measurable changes in glucose, triglycerides, electrolytes, and several blood cell parameters.³ Some analytes are especially sensitive to meal timing. A bone turnover marker called serum CTx, for instance, fluctuates by roughly 40% over a 24-hour cycle, peaking around 5 a.m. and bottoming out around 2 p.m. Fasting reduces this swing to about one-quarter of its usual magnitude, which is why samples for this marker are collected in the fasting state.⁴

Step One: Blood Collection

A phlebotomist draws blood from a vein, typically in the inner elbow, using a vacuum-sealed tube called a vacutainer. The type of tube used is critical. For serum, the tube contains no anticoagulant. Instead, many serum tubes include a clot activator to speed up the clotting process. Common clot activators include silica, kaolin, and thrombin. Silica and kaolin work by providing a surface that triggers the body’s natural clotting cascade, while thrombin directly converts fibrinogen into fibrin to form the clot.⁵

Many modern serum tubes also contain a gel separator, a layer of inert polymer that sits at the bottom of the tube. During centrifugation, this gel migrates upward to form a physical barrier between the serum above and the clotted blood cells below. The barrier helps keep the serum clean and makes it easier to pipette off without disturbing the clot. However, gel separators are not completely inert for all purposes. After 72 hours of room-temperature storage, certain drugs like verapamil and lercanidipine showed concentrations roughly 43% and 29% lower in gel tubes compared to non-gel tubes, suggesting the gel can absorb some compounds over time.⁶ For routine chemistry, this is rarely an issue, but therapeutic drug monitoring labs sometimes prefer non-gel tubes to avoid the risk.

Why the Order of Draw Matters

When a phlebotomist fills multiple tubes from the same needle stick, the sequence matters. Clinical guidelines recommend that citrate tubes for coagulation testing be drawn before serum tubes containing clot activators. The concern is that trace amounts of clot activator from the serum tube could carry over into subsequent tubes through the needle and affect coagulation test results.⁷ Research has found that when standard phlebotomy procedures are followed, the actual impact of this carryover on most coagulation tests is minimal, though thrombin-containing tubes can cause a noticeable change in one marker called fibrin monomer complex.⁸ In practice, labs stick to the recommended draw order to eliminate even small risks of cross-contamination.

Step Two: Clotting

Once the blood is in the tube, it needs time to clot. The tube is placed upright, and the blood sits at room temperature while fibrinogen converts to fibrin and forms a solid clot that traps the blood cells. Most protocols call for 30 to 60 minutes of clotting time.¹

This waiting period might seem like an area where cutting corners could save time, but rushing the clot risks incomplete clotting. If you centrifuge blood before the clot has fully formed, residual fibrin strands can end up in the serum and interfere with testing instruments. On the other hand, leaving the blood sitting too long before separation introduces its own problems: substances leak out of cells into the surrounding serum, changing the measured concentrations. One study tracking how serum-clot contact time affects lab results tested separation at 30 minutes, 3 hours, 6 hours, and 24 hours to quantify these shifts.⁹

So does it matter whether you wait 30 minutes or 60 minutes? For at least some routine analytes, no. A study comparing total cholesterol results after 30 and 60 minutes of clotting found a mean difference of about 3%, which was not statistically significant.¹⁰ The practical takeaway: anywhere in that 30-to-60-minute window tends to work for common tests, though specific assays may have tighter requirements.

Step Three: Centrifugation

After the clot has formed, the tube goes into a centrifuge, which spins it at high speed. The spinning force pushes the heavier clot and any remaining cells to the bottom, leaving the lighter serum on top. If the tube has a gel separator, the gel repositions itself between these two layers during the spin.

Standard protocols typically spin at around 1,000 to 2,000 times the force of gravity (expressed as ×g) for 10 to 15 minutes. But labs have explored whether higher speeds for shorter durations produce equivalent results. One study compared a 5-minute spin at 3,000 ×g against a standard 10-minute spin at 2,200 ×g and found that the faster protocol worked for most general chemistry and immunoassay analytes, with the possible exception of lactate dehydrogenase, which needed further assessment.¹¹ This kind of optimization matters in busy hospital labs where a few minutes saved per sample adds up across hundreds of daily specimens.

Spinning too aggressively or for too long can cause hemolysis, the rupturing of red blood cells. This releases hemoglobin and intracellular contents into the serum, visibly tinting it pink or red and interfering with many assays.¹² Getting centrifugation right is a balance between fully separating the serum and not destroying cells in the process.

Step Four: Separation and Aliquoting

Once centrifugation is done, the serum is carefully removed from the tube. In gel-separator tubes, this is straightforward because the gel barrier prevents accidental disturbance of the clot. A technician uses a pipette to transfer the clear serum into smaller labeled vials called aliquots. Dividing the serum into multiple aliquots is important for storage purposes, since each aliquot can be thawed and tested independently without exposing the rest to temperature changes.

At this point, the serum should be a clear, pale yellow liquid. Any cloudiness, redness, or unusual color signals a potential quality issue that the lab needs to evaluate before testing.

What Can Go Wrong: Hemolysis, Lipemia, and Icterus

The three classic interference flags in serum are hemolysis, lipemia, and icterus. Each discolors the sample and can distort test results in different ways.

Hemolysis, the most common pre-analytical problem in clinical labs, turns serum pink or red.¹³ It can happen at nearly any step: a difficult blood draw, a needle that is too small, excessive shaking of the tube, rough transport, or overly aggressive centrifugation. Even the method of transport within a hospital matters. Pneumatic tube systems, which shoot specimen carriers through building-wide tubing at high speed, have been associated with more hemolysis than hand-carried samples, though whether the difference is clinically meaningful is debated.¹²

Lipemia is milky cloudiness caused by high levels of triglyceride-rich particles in the blood, usually after a fatty meal. These particles scatter light, which is a problem because many lab instruments measure analytes by shining light through the sample and detecting how much passes through. A lipemic sample can therefore throw off absorbance-based assays. The interference is dose-dependent: the more triglycerides present, the greater the distortion.¹⁴ Lipemia can also block antibody binding sites in immunoassays, producing either falsely high or falsely low results depending on the specific test.¹⁵

Icterus, a yellow-orange discoloration from elevated bilirubin, is the third member of this trio. Each of these interference types is mechanistically distinct. Modern analyzers often automatically flag samples for hemolysis, lipemia, and icterus indices so that lab personnel can decide whether results are reliable or whether the sample needs to be recollected.

Storing Serum Without Degrading It

How serum is stored after separation determines how long it stays useful. For testing within a few hours, refrigeration at 2-8°C is standard. For longer-term storage, freezing is essential, and the temperature chosen makes a real difference.

A comparison of serum stored at −20°C versus −80°C found that out of 193 analytes checked, 12 showed markedly different concentrations depending on which temperature was used. Eight analytes, including the amino acids methionine and glutamine, were higher at −80°C, while four, including glutamate, were lower. Most analytes, about 120 out of 193, were robust to the temperature difference.¹⁶ For research biobanks that archive samples for years, −80°C is the standard choice.

Freeze-thaw cycles are another concern. Every time a frozen sample is thawed and refrozen, some proteins degrade or aggregate. One study found that roughly 14% of identifiable serum proteins showed a statistically significant intensity change after just a single freeze-thaw cycle.¹⁷ Repeated freeze-thaw cycles compound the problem, though the effect varies by analyte. Some markers like interferon-γ hold up well through five cycles, while others like VEGF and MMP-7 steadily climb in concentration with each cycle. Serum appears to handle repeated freezing somewhat better than plasma for circulating proteins, with one study showing increases of about 7% in serum versus roughly 15% in plasma for affected markers.¹⁸ This is why labs divide serum into small aliquots: you thaw only what you need, and the rest stays frozen.

Serum Beyond Diagnostics: Therapeutic Eye Drops

Serum is not just for lab testing. One of its more surprising applications is as an eye drop. Autologous serum eye drops, made from a patient’s own blood, are used to treat severe dry eye and certain corneal surface disorders. The idea is that serum contains growth factors, vitamins, and anti-inflammatory proteins that closely resemble the composition of natural tears, making it a more biologically appropriate lubricant than artificial tear products.

The preparation process follows the same basic steps described above: the patient’s blood is drawn, allowed to clot, centrifuged, and the serum separated. It is then diluted (typically to 20% concentration with saline), divided into small dropper bottles, and frozen until use. Strict protocols for preparation and storage have been shown to keep these drops free of bacterial contamination even in hospital settings.¹⁹ Research into optimizing these drops has explored whether the patient needs to be fasting when the blood is drawn. The fasting state makes the serum easier to filter (less lipid content), but the concentrations of the growth factors and lipids that make the drops therapeutic do not appear to change meaningfully based on fasting status.²⁰

Fetal Bovine Serum in Research and Cell Culture

The serum most widely used in biological research is not human at all. Fetal bovine serum (FBS) is a staple of cell culture, providing the nutrients, hormones, and growth factors that cells need to survive and divide in a lab dish. Its production follows the same clotting-and-centrifugation logic, but the source is very different.

FBS is typically obtained from the fetuses of pregnant cows at slaughterhouses. The pregnancy is usually unintended, discovered only after slaughter. In government-approved facilities, blood is collected from the fetus under sterile conditions, stored in sterile containers, and refrigerated to promote coagulation. The resulting clot is then separated by centrifugation in a refrigerator to yield the clear yellow serum, which is free of cellular components and clotting factors.²¹ The ethical concerns around FBS production have driven substantial research into serum-free and chemically defined cell culture media as alternatives, though FBS remains widely used because no single substitute yet matches its broad effectiveness across different cell types.

Miniaturizing the Process: Microfluidic Separation

Traditional serum preparation requires a lab, a centrifuge, and a trained technician. Researchers are working on shrinking the whole process down to a chip small enough to hold in your hand. Microfluidic devices use tiny channels etched into a plastic or silicone chip to separate blood components based on size. One such device demonstrated the ability to extract more than 0.1 microliters of plasma from a single droplet of about 5 microliters of whole blood, achieving over 98% purity in 3 to 5 minutes with no external power source at all.²²

These devices currently separate plasma rather than serum (since the blood does not have time to clot in the chip), but the underlying principle of separating the liquid fraction from the cellular fraction without a centrifuge is the same. The volumes produced are tiny, enough for a specific diagnostic test but not for a full chemistry panel. The long-term goal is a disposable chip that a patient could use at home for a fingerstick blood test, with results read by a smartphone or a simple optical reader. That technology is still in development, but the fact that high-purity separation can happen passively on a device smaller than a credit card suggests the bulky centrifuge may eventually become optional for certain applications.