If you are looking to source blood products for your life science research, choosing the right anticoagulant can be daunting. In this blog, we will provide an overview of the most used anticoagulants, their applications, advantages and disadvantages.
So, let’s start with the basics…
How do anticoagulants work?
Anticoagulation occurs in the two following ways:
- Binding calcium ions (e.g. EDTA, citrate)
- Inhibiting thrombin activity (e.g. heparin)
The three most used anticoagulants are:
- Ethylenediaminetetraacetic acid (EDTA)
- Heparin
- Citrate
How can anticoagulants interfere with analysis?
The coagulation process can change the concentrations of various constituents in extracellular fluid, pushing them beyond their maximum allowable limits. To obtain clinically relevant results, certain constituents should only be measured in plasma, such as ammonia, serotonin and neuron-specific enolase.
Therefore, adding anticoagulants can interfere with certain analytical methods or alter the concentrations of the constituents to be measured, such as:
- Contamination with cations: NH4+, Li+, Na+, K+
- Assay interference: Caused by metals complexing with EDTA and citrate, for example:
- Inhibition of alkaline phosphatase activity by zinc binding
- Inhibition of metalloproteinases
- Inhibition of metal-dependent cell activation in function tests
- Binding of ionised calcium to heparin
- Interference by fibrinogen: In heterogeneous immunoassays
- Inhibition of metabolic or catalytic reactions by heparin: For example, Taq polymerase in the polymerase chain reaction (PCR)
- Interference in ion distribution: Between the intracellular and extracellular spaces, such as Cl-, NH4+ by EDTA and citrate
Can anticoagulants affect PCR assays?
Yes. Anticoagulants, more specifically those that are heparin-based, can inhibit downstream PCR assays. PCR analysis can be applied to a wide variety of biospecimens, and the following precautions must be taken when using heparinised material where other anticoagulants cannot be used.
- Simple PCR tests: For simple PCR tests which do not require high sensitivity, dilution of the prepared nucleic acids is usually sufficient to overcome the inhibition. If heparinised material must be used and a more sensitive DNA PCR is required, nucleated cells should be isolated first, then washed repeatedly in physiological buffers before further processing
- Highly sensitive RT-PCR methods: For highly sensitive RT-PCR methods, additional measures are necessary to overcome heparin inhibition. Less effective methods include boiling, Sephadex chromatography, pH shifts with subsequent gel filtration, repeated ethanol precipitations, and treatment with protamine sulphate. Although treatment with heparinase restores amplification, this enzymatic purification step is costly. Additionally, RNA may be degraded during enzyme incubation by traces of RNase still present in the sample or by heparinase preparations contaminated with RNase
- Lithium chloride method: Recently, it has been demonstrated that lithium chloride can separate heparin from RNA, thus reversing the inhibition. This method, which reliably restores amplification from heparinised blood samples, is easily incorporated into a routine RNA preparation procedure without additional effort and so would be the preferred method
Anticoagulant selection guide
The following table includes commonly used anticoagulants, appropriate applications, and why:
- If you are using a mobile device, scroll left and right to view all columns
|
Anticoagulant |
Most suitable applications |
Not recommended for |
Advantages |
Disadvantages |
|
K2EDTA / K3EDTA |
|
|
|
|
|
Lithium/sodium heparin |
|
|
|
|
|
Sodium citrate |
|
|
|
|
|
ACD-A |
|
|
|
|
|
CPD |
|
|
|
|
|
Fluoride / oxalate (mixture of potassium oxalate & sodium fluoride) |
|
|
|
|
When to choose serum for your research application: metabolomics studies
For the reasons mentioned earlier, serum (the portion of plasma remaining after blood clotting in the absence of any anticoagulants) is recommended for metabolomics studies to avoid interference from anticoagulants in downstream applications. Serum is widely used for serological diagnosis of infectious diseases using multiple techniques including immunodiffusion, immunoprecipitation, counter immunoelectrophoresis, bacterial agglutination, haemagglutination and agglutination inhibition, particle-enhanced agglutination, complement fixation, indirect immunofluorescence (IFA), enzyme-linked immunoassay (ELISA), radioimmunoassay (RIA), neutralisation of toxins or virus activity, immunoblot (Western blot), etc.
For other tests, including some haemagglutination tests, ELISAs, or immunoblots, either serum or plasma may be used.
If serum is unavailable, both heparin plasma and EDTA plasma approximate the concentrations observed in serum closely.
Considerations for using gel separator tubes in metabolomics
An important distinction needs to be made between conventional blood collection tubes and gel separator tubes for metabolomic analysis. Gel separator tubes are used to accelerate the process of serum or plasma separation and due to the inert gel used, should not change the metabolite composition. However, several studies have highlighted differences in the metabolite fingerprints of samples collected using gel tubes compared to conventional tubes, particularly for amino acids. Hence, the use of gel separator tubes is not recommended.
Meeting your exact research project needs
We ensure you get the Right Donor, the Right Sample at the Right Time for your research. We collect from a large diverse pool of donors. Sample types include whole blood, leukopaks, PBMCs, buffy coat, plasma, serum and much more.
All samples are collected under a comprehensive informed consent for commercial and genetic research, and are processed to the highest quality standards in an ISO accredited and HTA (Human Tissue Authority) licenced facility
If you would like to talk to us about your requirements, please e-mail administration@researchdonors.co.uk or call +44 (0) 207 870 3742
References & further reading
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