Serum/Plasma Sample Collection and Preparation in Metabolomics

Blood contains more than 4000 endogenous small molecules metabolites, which can better reflect the overall level of the organism and is one of the commonly used samples for clinical studies and animal experiments. The metabolism in blood is dynamically regulated and its composition is constantly changing. Many unstable components may be oxidized, polymerized or degraded due to small changes in exogenous factors, so the pre-treatment process of blood samples can be relatively complex. While taking care to minimize individual differences between samples, it is necessary to ensure reproducibility of the experiments and to prevent significant dynamic changes in blood metabolites in vitro.

The process of selecting, collecting and processing biological samples is the most critical step in an experiment and directly determines the accuracy of the experimental results. Different processing procedures for the same sample can make the results significantly different and even lead to extremely biased conclusions. Therefore, unifying and standardizing blood sample processing procedures can reduce errors.

Selection of Serum or Plasma

Serum and plasma are commonly used blood-derived samples. Both, although similar in composition and properties, produce different findings as biological samples in metabolomics. In contrast to plasma, serum requires 30-60 min of agglutination, during which activated platelets may release a variety of compounds (e.g., proteins, lipids, etc.) that affect the metabolic composition of serum. Therefore, it is important to ensure that each sample has the same clotting time when using serum. The stability and reproducibility of plasma samples are good. However, anticoagulants can cause matrix effects thus affecting the assay results.

Processing and Storage of Blood Samples

Hemolysis is one of the major risks in both clinical studies and animal experiments. The causes of hemolysis include rapid blood sampling, strong shaking of blood collection tubes, high centrifugation rates, and inappropriate storage temperatures. Hemolysis leads to the release of intracellular compounds (e.g., metabolites, enzymes, etc.), which can cause changes in the metabolites of the blood sample. Changes in blood clotting time and ambient temperature can also affect metabolism. This may be due to continuous release of metabolites from blood cells or decay of released metabolites. Therefore, collectors should collect blood in anticoagulation tubes if using plasma samples. Centrifuge at low temperature (4°C) (2000 × g, 20 min) and remove the supernatant for storage. If the actual collection of clinical samples cannot be centrifuged in time, they can be transferred with an ice box and violent shaking should be avoided during the process. If serum samples are used, blood should be collected in a centrifuge tube and left at room temperature for 30-60 min, followed by centrifugation at low temperature (4°C) (2000×g, 10 min) and storage of the supernatant.

Cryopreservation is the next step in sample processing and direct storage at -80°C is the preferred condition. If clinical conditions are limited, samples may be stored at -20°C before being transferred on dry ice. Some metabolite concentrations (e.g., glucose and proline) can change significantly after 1 month of plasma storage at -20°C. It is recommended that blood samples be stored frozen at -80°C in a refrigerator and placed vertically to avoid contamination of the sample cap during thawing and analysis.

Freeze-thawing of Blood Samples

Frozen and thawed serum and plasma from blood samples stored at -80 °C are relatively stable. Repeated freeze-thawing may be required due to experimental requirements. It is recommended that samples be placed in a 4 °C environment for gradual thawing. Some metabolites may change significantly or lose stability after 4 to 5 freeze-thaw cycles at room temperature. Therefore, to avoid the potential effects of repeated freeze-thawing, it is recommended that animal samples be packed in 0.2 mL/tube and clinical samples in 0.4 mL/tube into centrifuge tubes or lyophilization tubes for freezing and storage.

Effects of Freeze-Thaw Cycles (Chen et al., 2020)

Reference

1. Chen, D., Han, W., et al. (2020). Effects of Freeze–Thaw Cycles of Blood Samples on High-Coverage Quantitative Metabolomics. Analytical Chemistry, 92(13), 9265-9272.