Peripheral blood is one of the commonly used samples in our flow experiments. Since blood coagulates in the presence of coagulation factors after it is isolated from the body, the collection of peripheral blood samples requires the use of blood collection tubes with anticoagulants or the addition of anticoagulants immediately after collection. Typically, there are two types of anticoagulants used in our flow experiments: EDTA and heparin. Translated with DeepL.com (free version)
1.Principle of anticoagulation of two anticoagulants
EDTA (ethylenediaminetetraacetic acid):
EDTA can chelate with Ca2+ in the blood, making Ca2+ lose its coagulation effect and preventing the blood from coagulating. Sodium or potassium salts are commonly used.
Heparin:
Enhances the action of antithrombin (AT) to inactivate serine protease, preventing the formation of thrombin and platelet aggregation, thus preventing blood clotting.
2.Characteristics of the two anticoagulants
EDTA
Potassium salts have better solubility in blood and are therefore used more often, especially the dipotassium salt - EDTA-K2 can be attached to the inner surface of blood collection tubes by spray-drying and does not dilute the collected blood samples.
EDTA has a relatively small effect on blood cell morphology and has good stability, but EDTA inhibits or interferes with the polymerization of fibrin monomers in the formation of fibrin clots, making it unsuitable for blood coagulation and platelet function assays, as well as for the determination of calcium, potassium, sodium, and nitrogen-containing substances.
Heparin
Heparin has a low chelating power, little effect on the movement of water molecules, little interference with blood components, does not affect the volume of red blood cells, and does not cause hemolysis, which can be considered to have many advantages. However, heparin also has two distinct disadvantages:
● Heparin is mainly isolated from animal tissues such as pigs, cows and sheep, and is more costly compared to other anticoagulants;
● Heparin can only slow down the clotting process but not stop it. In practice, it has been found that whole blood using sodium heparin as an anticoagulant gradually develops clotting clumps at room temperature for 10 to 12 hours.
3.EDTA or Heparin
Since the two anticoagulants will interfere with the blood in different ways, we usually choose the appropriate anticoagulant according to the purpose of the experiment.
a.If samples are lysed directly from red blood cells for surface indicator staining experiments, both anticoagulants can be used;
b.Because heparin will affect platelets, heparin is not recommended for all platelet-related experiments;
c.EDTA anticoagulation is not recommended if blood is collected for cytokine testing.
After anticoagulation with both anticoagulants, cytokines were detected, and IFN-γ expression was significantly lower in EDTA-anticoagulated samples than in heparin-anticoagulated samples.
Since EDTA can chelate with Ca2+, resulting in a decrease in Ca2+ transported by the cytokine stimulator, ionomycin (Ionomycin), which is essential for cytokine secretion, heparin anticoagulation is recommended for cytokine testing.
Regardless of which anticoagulant is used, it is important to allow the anticoagulant to mix well with the blood after collection to achieve anticoagulation.
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