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【Technology Sharing】Preparation of Serum and Plasma from Blood

1. Serum and Plasma Preparation

Preparation of serum and plasma from blood sample detected with ELISA is specified below.

1.1. Serum Preparation from Blood

  • Let the whole blood sample collected from serum separator tube stand for 2 hours at room temperature or overnight at 2-8℃. (This is a natural blood coagulation procedure. The coagulation process is slower with the drop of temperature. Blood clotting accelerant can be added according to assay requirements.);
  • Centrifuge the sample for 20 minutes at 1000×g and collect the supernatant;
  • Perform the assay immediately or sub-pack for cryopreservation at -20℃ or -80℃.

1.2. Plasma Preparation from Blood (Citric Acid, EDTA, Heparin)

  • Plasma sample requires for anticoagulation. Collect the whole blood into tube containing anticoagulant and mix gently;
  • Centrifuge the sample for 15 minutes at 1000×g at 2-8℃ within 30 min of collection;
  • Perform the assay immediately or sub-pack for cryopreservation at -20℃ or -80℃.

2. Anticoagulant Types and Uses

Select different anticoagulants suitable for the property of target samples to be tested.

2.1. Citrate(Sodium Citrate)

Ca2+ has the coagulant effect. Citrate can form soluble chelate with Ca2+ in the blood to prevent blood coagulation. The general anticoagulant working concentration of citrate is 0.25%. (The proportion between anticoagulant and blood is 1:16 when using 4% anticoagulant.)
Advantages: Protect coagulation factor well. Most coagulation tests can use the citrate for anticoagulation.
Disadvantages: Low solubility in the blood, weak anticoagulation.

2.2. Ethylenediamine Tetraacetic Acid (EDTA)

Form coordination compounds by binding with Ca2+ in the blood to prevent blood coagulation. The anticoagulant working concentration of EDTA: 10ml blood contains 12mg EDTA.
Advantages: Small influence on erythrocyte and leukocyte morphology.
Disadvantages: Interference with platelet aggregation. Unsuitable for coagulation tests and platelet functional detection.

2.3. Heparin

Enhance effects of antithrombin by binding with antithrombin Ⅲ(AT-Ⅲ). Inactivate serine protease to prevent the formation of thrombin, platelet aggregation and blood coagulation. Pure 10mg heparin the anticoagulant effect in 65~125 ml blood.
Advantages: Strong anticoagulation, no effects on blood cell volume, difficult hemolysis, thermotolerant.
Disadvantages: Cause leucocyte aggregation. Heparin anticoagulant should be used in a short time. Otherwise, blood will be coagulated again after a longer time.

3. Selection and Differences between Serum and Plasma

  • Serum is the plasma without fibrinogen after the blood is coagulated. Thus, except fibrinogen and anticoagulant, other components of plasma are equivalent to serum;
  • Plasma sample is recommended to detect coagulation factors due to lack of many coagulation factors in the serum containing coagulation products;
  • Serum sample is recommended if fibrinogen in the plasma has an effect on the target to be tested;
  • In most situations, both serum and plasma can be selected.


[1] Blood Sampling and Preparation Procedures for Proteomic Biomarker Studies of Psychiatric Disorders, PMID: 28353230.
[2] Neuro biomarker levels measured with high-sensitivity digital ELISA differ between serum and plasma, PMID: 31829739.
[3] Sample collection in clinical proteomics--proteolytic activity profile of serum and plasma, PMID: 24723329.