Which anticoagulant should I choose?

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

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

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.