A researcher's guide to human blood: which anticoagulant should I choose?

For someone who is new to research studies involving blood products, choosing the right anticoagulant can be daunting. In this blog, we will provide you with an overview of the most commonly used anticoagulants, highlighting their compatible applications, advantages and disadvantages. By the end, you’ll have helpful insights and clarity on the best option for your work.

So, let's start with the basics...

How do anticoagulants work?

Anticoagulation occurs by either binding calcium ions (e.g. EDTA, citrate) or inhibiting thrombin activity (e.g. heparain). The most commonly used anticoagulants are ethylenediaminetetraacetic acid (EDTA), heparin, and citrate.

How can anticoagulants interfere with analysis?

The coagulation process can alter the concentrations of numerous constituents in the extracellular fluid, pushing them beyond their maximum allowable limits. To obtain clinically relevant results, certain constituents should only be measured in plasma, such as neuron-specific enolase, serotonin, and ammonia.

The addition of anticoagulants can, therefore, 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

How can anticoagulants affect PCR assays?

A fundamental mention should be made of the inhibition that anticoagulants, more specifically heparinates, can have on downstream PCR assays. PCR analysis can be applied to a wide variety of materials, including whole blood or bone marrow containing an anticoagulant such as EDTA or citrate, serum or plasma, dried blood (filter-paper cards), buffy coat, sputum, mouthwash, bronchial lavage, cerebrospinal fluid, urine, stool, biopsy material, cell cultures, fixed tissue, embedded tissue, tissue sections, and more. Because heparin inhibits PCR reactions, specific precautions must be taken when using heparinised material if other anticoagulants cannot be used.

For heparinised specimens, the following considerations must be taken into account:

1. Simple PCR tests: For simple PCR tests not requiring 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 and washed repeatedly in physiological buffers before further processing.
    
2. Highly sensitive RT-PCR methods: For highly sensitive RT-PCR methods, additional measures are necessary to overcome heparin inhibition. Methods that have failed include boiling, Sephadex chromatography, pH shifts with subsequent gel filtration, repeated ethanol precipitations, and treatment with protamine sulfate. 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.
    
3. 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.

Anticoagulant selection guide

The table below includes the anticoagulants that are routinely used, the applications they are most appropriate for, and the reasoning behind their use:

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 infereance from anticoagulants in downstream applications. Generally, serum is used for serological diagnosis of infectious diseases using methods such as 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), and others.

Serum must be used for certain immunological techniques such as complement fixation or bacterial agglutination tests. 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 those utilising gel separator tubes for investigators considering metabolomics analysis. Gel Gel separator tubes are used to accelerate the process of serum or plasma separation and theoretically should not change the metabolite composition because of the inertness of the gel. However, several studies have shown changes in the metabolite fingerprints of samples collected using polymeric gel tubes compared to conventional tubes, particularly for amino acids. As such, the use of gel separator tubes is not recommended.

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