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.
REFERENCES
[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.