Plasma sample preparation methods

In the fields of biomedical research, drug analysis, and clinical diagnosis, plasma samples play a vital role as analysis objects. Plasma, as the liquid part of blood, is rich in various biological molecules, including proteins, lipids, metabolites, and possible drug residues. However, these abundant components also present analytical challenges, as many of them may act as interfering factors, affecting the accurate determination of target analytes. Therefore, the pretreatment of plasma samples has become a key link to ensure the reliability and accuracy of experimental results.

1. General processing methods 1. Anticoagulation to collect plasma: After collecting anticoagulated whole blood, gently invert it to ensure sufficient anticoagulation, and then directly centrifuge at low speed to separate the plasma (you can also let it sit for about half an hour and then centrifuge). Appropriate centrifugal force and time should be selected during centrifugation to avoid hemolysis. Commonly used anticoagulants in laboratories include various salts of heparin, EDTA and sodium citrate. The appropriate anticoagulant needs to be selected according to experimental needs. 2. Low-temperature cryopreservation: The separated plasma can be frozen and stored, but if flocculent turbidity appears after thawing, it should be removed by centrifugation before measurement.

2. Methods for removing interfering substances 1. Dilution heating method: The plasma is diluted 3 to 10 times with dissolved water, and then heated in 37 to 100°C water. The holding time is generally 5 to 10 minutes. This method can remove or reduce the content of certain heat-sensitive interfering substances. 2. Chloroform extraction method: add an equal amount of chloroform to the plasma and vibrate for a period of time (such as 3 hours), and then take the upper layer for detection. Chloroform can effectively extract and separate certain fat-soluble interfering substances. 3. Ether treatment method: The plasma is treated with ether and insulated at an appropriate temperature (such as 40°C), and then the ether is removed. Ether can remove or reduce certain volatile interfering substances in plasma. 4. Trifluoroacetic acid method: Add acetic acid to the plasma to adjust the pH to 4.0, and then use K2HPO4 to adjust the pH to 7~7.5. This method can change the acid-base environment of plasma, thereby affecting the stability and activity of certain interfering substances. 5. Surfactant treatment method: similar to the dilution heating method, but the dilution water is changed to surfactant. Surfactants can destroy certain membrane structures in plasma and release interfering substances wrapped in membranes. 6. Gel filtration method: Use the molecular sieve function of gel to separate substances of different molecular sizes in plasma. This method can effectively remove macromolecular interfering substances. 7. Alkaline treatment method: The plasma is incubated with a certain concentration of alkali solution (such as 0.2N NaOH) at an appropriate temperature (such as 37°C) for a period of time (such as 10 minutes), and then neutralized with an acid solution (such as HCl). An alkaline environment can change the chemistry of some interfering substances, making them easier to remove or reducing their activity. 8. Beads method: Put special resin that adsorbs specific interfering substances (such as resin that adsorbs endotoxin) into the plasma, take it out after it is fully adsorbed, and then add appropriate reagents for subsequent reactions. This method allows for the targeted removal of specific interfering substances in plasma.

3. Pretreatment methods for specific target substances If the pretreatment of plasma samples is to measure a specific target substance (such as weakly alkaline drugs), a more targeted pretreatment method may be required. For example: 1. Use trichloroacetic acid or perchloric acid to precipitate plasma proteins, and then take the supernatant for subsequent processing. 2. Adjust the pH value to weak alkalinity to free the target drug, and then add organic solvent for extraction and purification.

Plasma sample preprocessing is an indispensable part of biomedical research, drug analysis and clinical diagnosis. By using appropriate pretreatment methods, we can effectively remove or reduce interfering substances in plasma, protect and enrich target analytes, and thus provide high-quality samples for subsequent experimental analysis.