Researcher's human blood guide, which anticoagulant to choose?

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:

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Anticoagulant	Most suitable applications	Not recommended for	Advantages	Disadvantages
K2EDTA / K3EDTA	Whole blood haematology determinations, immunohematology & donor screening Downstream PCR Platelets counting Peripheral blood smear	PBMCs isolation Calcium and iron estimation	Less hyperosmolar effect on blood cells (K2EDTA is preferred) Preservation of morphology of the RBCs	Promotion of platelets’ adherence to neutrophils Inhibition of alkaline phosphatase, creatine kinase and leucine aminopeptidase activities Diluted specimen (liquid additive – K3EDTA only)
Lithium/sodium heparin	Plasma determinations Haematological tests pH, blood gases electrolytes & ionised calcium estimations	Downstream PCR Red blood cell preservation Proteomic studies Peripheral blood Smear	Preservation of morphology of the RBCs Minimal haemolysis	Inhibition of acid phosphatase activity
Sodium citrate	Coagulation studies Platelet function tests	Red blood cells preservation PCV, Hb, TLC and DLC tests Calcium estimation	Preserves coagulation factors Reversible anticoagulation	Inhibition of aminotransferase & alkaline phosphatase
ACD-A	Blood & tissue typing DNA analysis PBMC preservation Red blood cell preservation	Coagulation studies Biochemical analysis Metabolomics studies	Investigation of platelet function for longer periods of time (6-8hrs) Prolonged whole blood/RBC shelf-life (21 days) Stabilisation of lymphocytes for establishment of Lymphoblastoid cell lines (LCLs) Prevents haemolysis Maintains pH & preserves ATP levels	Impact on biochemical assays
CPD	PBMC preservation Red blood cell preservation		Prolonged whole blood/RBC shelf-life (28 days) Isotonicity for red blood cells Decreases acidosis and improves ATP synthesis Better maintenance of 2,3 DPG
Fluoride / oxalate *(mixture of potassium oxalate & sodium fluoride)*	Glucose determinations on plasma Whole blood haematology determinations Blood alcohol testing	Clinical chemistry tests & enzymatic immunoassays Peripheral blood smear	Preservation of glucose concentrations	Inhibition of many enzymes Interference with electrolyte measurements Morphology of the WBCs not preserved well

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

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