How to choose anticoagulant in bioanalytical sample collection?



In the process of biological analysis, the collection of blood samples is a very important link. Selecting appropriate anticoagulants and stabilizers can minimize interference, which is beneficial to the stability of drugs and their metabolites, and reduces the test caused by coagulation. The pressure and error in the operation are the prerequisites to ensure the stability and reliability of the biological analysis method.

What is the mechanism of action of anticoagulants?
The coagulation reaction is the conversion of fibrinogen into fibrin by thrombin, from which a fibrin clot is formed, in which thrombin is generated by the cleavage of prothrombin under the action of coagulation factors and calcium ions, and chelating anticoagulants are produced by Combining with calcium ions to block coagulation, the mechanism of action of heparin is more complicated, and its anticoagulant effect is mainly achieved by inhibiting the conversion of prothrombin into thrombin.

1. Ethylenediaminetetraacetic acid (EDTA)
1) Ethylenediaminetetraacetic acid (EDTA) is currently the most widely used and widely used anticoagulant. EDTA can chelate with any form of calcium ions to prevent blood coagulation and play an anticoagulant effect.
2) Working principle:

3) Features:
Both sodium and potassium salts are commonly used in different forms, but potassium salts are more soluble in blood and are used more. In addition, the physical form of K3EDTA is liquid at room temperature, while K2EDTA is characterized by spray drying. The method makes it adhere to the inner surface of the blood collection tube, so it will not dilute the collected blood sample, so the form of dipotassium salt is more widely used in practice. Compared with heparin sodium blood collection tubes, blood samples collected with EDTA as an anticoagulant produce fewer clots after freezing and thawing, and the anticoagulation time in whole blood is longer, which is more conducive to subsequent experimental operations.

2. Heparin
1) Heparin is a high sulfated glycosaminoglycan, which is mainly composed of many disaccharide groups that can be sulfated. The commonly used molecular weight is 12~15kDa. The most commonly used salt as an anticoagulant is lithium. Sodium, Potassium Salts,

2) Principle: Heparin can play an anticoagulant effect from three different aspects. First, it can inhibit the conversion of prothrombin into thrombin, inhibit fibrin fibrosis, and stabilize platelets.
3) Features: Heparin has two disadvantages as an anticoagulant. First, heparin is mainly isolated from animal tissues such as pigs, cattle, and sheep. Compared with other anticoagulants, the cost is higher. It cannot prevent coagulation. In actual test use, it is found that whole blood using heparin sodium as an anticoagulant will gradually appear coagulation and clotting at room temperature for 10 to 12 hours.

3. Oxalates
1) As an anticoagulant with a simple structure, oxalate is commonly used in the form of oxalate such as ammonium, lithium, potassium, and sodium. Usually, ammonium oxalate and potassium oxalate are mixed in a ratio of 3:2. Also known as Paul Heller bis-oxalate.
2) Principle: Its mechanism of action is to combine with calcium ions to form insoluble calcium oxalate to inhibit blood coagulation.
3) Features: ①Oxalate as an anticoagulant will have the shortcomings of red blood cell dehydration (potassium oxalate) and swelling (ammonium oxalate), compared with heparin in whole blood samples, it will reduce hematocrit and cause plasma dilution effect, so Errors can occur when determining the concentration of a sample, so the Paul Heller bis-oxalate form is often used to reduce the effect of red blood cell deformation on plasma concentrations. ②Oxalic acid is a strong organic acid, and its salt form will change the pH of plasma, which may cause stability problems for some drugs that are sensitive to pH, but oxalate has an inhibitory effect on phosphatase, which can reduce the phosphate group The degradation of the group helps to stabilize phosphatase-sensitive drugs.

4. Citrates
1) Citric acid is one of the earliest anticoagulants used, usually 3.2% or 3.8% sodium citrate dihydrate.
2) Principle: It acts as an anticoagulant by chelating calcium ions in the blood to form calcium citrate.
3) Features: ①Citrate can act as a buffer in whole blood, which may change the pH of whole blood. For pH-sensitive drugs, attention should be paid to its stability. ②The biggest disadvantage is that compared with oxalate, it has more serious atrophy of red blood cells, resulting in the dilution of plasma, and the detected drug concentration is lower than the true value. Therefore, in the process of using 3.2% or 3.8% sodium citrate, it must be mixed with whole blood in strict accordance with the proportion to avoid dilution effect.

5. Fluoride
1) Fluoride is an anticoagulant with weak anticoagulant ability, and sodium fluoride is mainly used.
2) Principle: It acts as an anticoagulant by combining calcium ions to form calcium fluoride.
3) Features: ① Sodium fluoride has a strong enzyme inhibitory effect, which can effectively inhibit the activities of phosphatase, acetylcholinesterase and butyrylcholinesterase, and is often used as an effective stabilizer. The lower the value, the better the inhibitory effect. In actual use, it is usually used in combination with potassium oxalate (sodium fluoride-potassium oxalate). This combination can ensure effective anticoagulant effect while inhibiting more enzymes. ②Due to the poor solubility of sodium fluoride, 2% sodium fluoride solution is usually added into whole blood. This is the same as sodium citrate. It needs to be added and mixed in strict proportions and the possible dilution effect should be considered when using it.

6. Other auxiliary additives
1) Various compound stability problems will occur during the method development stage, which requires adding various additives to stabilize the compound during the blood collection process to avoid possible redox, enzymatic decomposition, chiral conversion and other degradation reactions. Specific adsorption (NBS) is possible to ensure that the developed bioanalytical method is robust and reliable.
2) Type: ①Antioxidant ②Enzyme inhibitor ③PH regulator ④Enzymatic reaction terminator ⑤Anti-adsorbent
Antioxidants: Commonly used are vitamin C, sodium metabisulfite and 2,6-di-tert-butyl-4-methylphenol (BHT)
Enzyme inhibitors: sodium fluoride (choline/carboxylesterase), dichlorvos (cholinesterase), phenylmethylsulfonyl fluoride (carboxylesterase), etc.

PH regulators: The most commonly used are phosphoric acid and phosphate buffered salts.
Enzymatic reaction terminator: organic reagents such as acetonitrile, methanol, etc.
Anti-adsorbents: surfactants such as Tween-20/80, sodium dodecylbenzenesulfonyl (SDBS), bovine serum albumin (BSA), β-cyclodextrin, etc.