A researcher’s guide to choosing the right anticoagulant for blood research, including PBMCs and plasma
Sourcing blood products for life science research can be daunting, especially when selecting the right anticoagulant for your study. Whether you’re working with PBMCs, plasma, serum, or whole blood, choosing the correct anticoagulant is crucial to avoid interference in assays and to preserve sample integrity.
What are anticoagulants and how do they work?
Anticoagulants prevent blood from clotting in two primary ways:
- Binding calcium ions – e.g., EDTA, citrate.
- Inhibiting thrombin activity – e.g., heparin.
The three most widely used anticoagulants are:
- Ethylenediaminetetraacetic acid (EDTA)
- Heparin
- Citrate
Potential interference of anticoagulants with analysis
While anticoagulants are essential in research, they can interfere with analytical methods or alter the concentrations of certain constituents in blood. Here are key considerations:
- Contamination with cations: For example, NH4+, Li+, Na+, and K+.
- Assay interference: Caused by the complexation of metals with EDTA and citrate, leading to:
- Inhibition of alkaline phosphatase and metalloproteinase activities.
- Binding of ionized calcium to heparin.
- Impact on fibrinogen: Can affect heterogeneous immunoassays.
- Inhibition of metabolic reactions: Heparin, for example, can inhibit Taq polymerase in PCR.
Can anticoagulants affect PCR assays?
Yes, particularly heparin-based anticoagulants, which can inhibit PCR analysis. To mitigate these effects, consider these precautions:
Simple PCR tests:
- Dilution of nucleic acids often resolves inhibition.
- For high-sensitivity PCR, isolate nucleated cells and wash them repeatedly in physiological buffers.
Highly sensitive RT-PCR:
- More advanced methods, such as lithium chloride treatment, effectively remove heparin without degrading RNA.
- Alternative techniques include Sephadex chromatography or heparinase treatment, but these can be costly and time-consuming.
Anticoagulant selection guide
The table below summarises the most common anticoagulants, their applications, advantages, and disadvantages:
|
Anticoagulant |
Best for |
Not recommended for |
Advantages |
Disadvantages |
|
K2EDTA / K3EDTA |
Haematology, donor screening, PBMC isolation |
Calcium/iron estimation, PCR |
Preserves RBC morphology, low hyperosmolar effect |
Can inhibit certain enzyme activities, diluted specimen (K3EDTA only) |
|
Lithium/sodium heparin |
Plasma determinations, pH and blood gas analysis |
PCR |
Minimal hemolysis, preserves RBC morphology |
Inhibits acid phosphatase, interferes with metabolic reactions |
|
Sodium citrate |
Coagulation studies, platelet function tests |
Calcium estimation |
Reversible anticoagulation, preserves coagulation factors |
Inhibits aminotransferase and alkaline phosphatase activities |
|
ACD-A |
PBMC preservation, DNA analysis |
Biochemical/metabolomics studies |
Prolongs shelf-life of blood (6–8 hours), stabilizes lymphocytes for LCL establishment |
Can impact biochemical assay results |
|
CPD |
PBMC preservation, red blood cell storage |
N/A |
Enhances ATP synthesis, isotonic for RBCs |
Limited compatibility with some analytical methods |
|
Fluoride/oxalate |
Glucose determinations, alcohol testing |
Enzymatic immunoassays, WBC morphology |
Preserves glucose concentrations |
Inhibits many enzymes, poorly preserves WBC morphology |
Serum vs. plasma for metabolomics studies
For metabolomics research, serum is often the preferred choice. Unlike plasma, serum avoids interference from anticoagulants, making it ideal for downstream applications such as:
- Immunoassays (ELISA, RIA).
- Serological diagnostics.
However, for some tests (e.g., ELISAs, immunoblots), both serum and plasma can be used interchangeably.
Considerations for gel separator tubes
While gel separator tubes are useful for rapid serum/plasma separation, studies show they may alter metabolite profiles, particularly for amino acids. To ensure accurate metabolomic analysis, avoid using gel separator tubes.
Ensuring quality for your research
At Research Donors, we provide high-quality blood products tailored to your research needs. Our extensive donor pool allows us to deliver diverse sample types, including whole blood, leukopaks, PBMCs, plasma, serum, and more.
- High standards: All samples are processed in an ISO-accredited, HTA-licensed facility under informed consent.
- Flexibility: Customise your sample requirements to suit your specific project.
Explore our selection of blood products and find the right sample type for your research. View our blood biospecimens to get started today!
References & further reading
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