Sales & Support: +44 (0)1223 316 855

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

Published 29 May 2024 by Eleonora Golfetto

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:

Anticoagulant Most suitable applications Not recommended for Advantages Disadvantages
  • Whole blood
    hematology 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


  • 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


  • 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 hematology 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 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.

We are here to help: Research Donors human blood service

We know that sourcing human blood for research can be challenging, complex and time consuming. Our complete human blood service, in partnership with Research Donors, offers you with fresh human blood and derivatives such as buffy coat, PBMCs, plasma and serum, as well as fresh human leukopaks from healthy volunteer donors. Our specialist blood team are here to help with any questions so we can help you source the right sample, from the right donor exactly when your research needs it. Learn more about our blood and biospecimens service or contact our blood specialists to discuss your needs today.

References & further reading

  • Betsou, F., Gaignaux, A., Ammerlaan, W., Norris, P.J. and Stone, M. (2019). Biospecimen Science of Blood for Peripheral Blood Mononuclear Cell (PBMC) Functional Applications. Current Pathobiology Reports, 7(2), pp.17–27. doi:https://doi.org/10.1007/s40139-019-00192-8.
  • Bowen, R.A.R. and Remaley, A.T. (2014). Interferences from blood collection tube components on clinical chemistry assays. Biochemia Medica, 24(1), pp.31–44. doi:https://doi.org/10.11613/bm.2014.006.
  • Caliezi, C., Reber, G., Lämmle, B., de Moerloose, P. and Wuillemin, W.A. (2000). Agreement of D-dimer results measured by a rapid ELISA (VIDAS) before and after storage during 24h or transportation of the original whole blood samples. Thrombosis and Haemostasis, [online] 83(1), pp.177–178. Available at: https://pubmed.ncbi.nlm.nih.gov/10669177/ [Accessed 29 May 2024].
  • Chowdhury, F.R., Rodman, H. and Bleicher, S. (1971). Glycerol-like contamination of commercial blood sampling tubes. Journal of Lipid Research, [online] 12(1), p.116. Available at: https://pubmed.ncbi.nlm.nih.gov/5542696/#:~:text=Abstract [Accessed 29 May 2024].
  • England, J.M., Rowan, R.M., van Assendelft, O.W., Bull, B.S., Coulter, W., Fujimoto, K., Groner, W., Richardson-Jones, A., Klee, G., Koepke, J.A., Lewis, S.M., McLaren, C.E., Shinton, N.K., Tatsumi, N. and Verwilghen, R.L. (1993). Recommendations of the International Council for Standardization in Haematology for Ethylenediaminetetraacetic Acid Anticoagulation of Blood for Blood Cell Counting and Sizing: International Council for Standardization in Haematology: Expert Panel on Cytometry. American Journal of Clinical Pathology, 100(4), pp.371–372. doi:https://doi.org/10.1093/ajcp/100.4.371.
  • Ladenson, J.H., L. M.B. Tsai, Michael, J.M., Kessler, G. and J. Heinrich Joist (1974). Serum versus Heparinized Plasma for Eighteen Common Chemistry Tests: Is Serum the Appropriate Specimen? Am J Clinical Pathology, 62(4), pp.545–552. doi:https://doi.org/10.1093/ajcp/62.4.545.
  • Leonard, P.J., Persaud, J. and Motwani, R. (1971). The estimation of plasma albumin by BCG RCG binding on the technicon SMA 12/60 analyser and a comparison with the HABA dye binding technique. Clinica Chimica Acta, 35(2), pp.409–412. doi:https://doi.org/10.1016/0009-8981(71)90214-2.
  • Li, G., Cabanero, M., Wang, Z., Wang, H., Huang, T., Alexis, H., Eid, I., Muth, G. and Pincus, M.R. (2013). Comparison of glucose determinations on blood samples collected in three types of tubes. Annals of Clinical and Laboratory Science, [online] 43(3), pp.278–284. Available at: https://pubmed.ncbi.nlm.nih.gov/23884222/ [Accessed 29 May 2024].
  • Meng, Q.H. and Krahn, J. (2008). Lithium heparinised blood-collection tubes give falsely low albumin results with an automated bromcresol green method in haemodialysis patients. Clinical Chemical Laboratory Medicine, 46(3). doi:https://doi.org/10.1515/cclm.2008.079.
  • Narayanan, S. (2000). The Preanalytic Phase. American Journal of Clinical Pathology, 113(3), pp.429–452. doi:https://doi.org/10.1309/c0nm-q7r0-ll2e-b3uy.
  • Neumaier, M., Braun, A. and Wagener, C. (1998). Fundamentals of quality assessment of molecular amplification methods in clinical diagnostics. International Federation of Clinical Chemistry Scientific Division Committee on Molecular Biology Techniques. Clinical Chemistry, [online] 44(1), pp.12–26. Available at: https://pubmed.ncbi.nlm.nih.gov/9550553/.
  • Numata, Y., Dohi, K., Furukawa, A., Kikuoka, S., Asada, H., Fukunaga, T., Taniguchi, Y., Sasakura, K., Tsuji, T., Inouye, K., Yoshimura, M., Itoh, H., Mukoyama, M., Yasue, H. and Nakao, K. (1998). Immunoradiometric assay for the N-terminal fragment of proatrial natriuretic peptide in human plasma. Clinical Chemistry, [online] 44(5), pp.1008–1013. Available at: https://pubmed.ncbi.nlm.nih.gov/9590374/ [Accessed 29 May 2024].
  • Peake, M.J., Bruns, D.E., Sacks, D.B. and Horvath, A.R. (2013). It’s Time for a Better Blood Collection Tube to Improve the Reliability of Glucose Results. Diabetes Care, [online] 36(1), pp.e2–e2. doi:https://doi.org/10.2337/dc12-1312.
  • Pignatelli, P., Pulcinelli, F.M., Ciatti, F., M. Pesciotti, Ferroni, P. and Gazzaniga, P.P. (1996). Effects of storage on in vitro platelet responses: Comparison of ACD and Na citrate anticoagulated samples. Journal of clinical laboratory analysis, 10(3), pp.134–139. doi:https://doi.org/10.1002/(sici)1098-2825(1996)10:3%3C134::aid-jcla4%3E3.0.co;2-b.
  • Rifai, N., Horvath, A.R. and Wittwer, C. (2018). Tietz textbook of clinical chemistry and molecular diagnostics. 6th ed. St. Louis, Missouri: Elsevier.
  • Sevastos, N., Theodossiades, G., Efstathiou, S., Papatheodoridis, G.V., Manesis, E. and Archimandritis, A.J. (2006). Pseudohyperkalemia in serum: the phenomenon and its clinical magnitude. The Journal of Laboratory and Clinical Medicine, [online] 147(3), pp.139–144. doi:https://doi.org/10.1016/j.lab.2005.11.008.
  • Sotelo-Orozco, J., Chen, S.-Y., Hertz-Picciotto, I. and Slupsky, C.M. (2021). A Comparison of Serum and Plasma Blood Collection Tubes for the Integration of Epidemiological and Metabolomics Data. Frontiers in Molecular Biosciences, 8. doi:https://doi.org/10.3389/fmolb.2021.682134.
  • Tammen, H., Schulte, I., Hess, R., Menzel, C., Kellmann, M., Mohring, T. and Schulz-Knappe, P. (2005). Peptidomic analysis of human blood specimens: Comparison between plasma specimens and serum by differential peptide display. PROTEOMICS, 5(13), pp.3414–3422. doi:https://doi.org/10.1002/pmic.200401219.
  • Tate, J. and Ward, G. (2004). Interferences in immunoassay. The Clinical biochemist. Reviews, [online] 25(2), pp.105–20. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1904417/#:~:text=Interference%20can%20lead%20to%20falsely.
  • Technology, W.H.O.D.I. and L. (2002). Use of anticoagulants in diagnostic laboratory investigations. iris.who.int. [online] Available at: https://iris.who.int/handle/10665/65957.
  • Toffaletti, J.G. and Wildermann, R.F. (2001). The effects of heparin anticoagulants and fill volume in blood gas syringes on ionized calcium and magnesium measurements. Clinica Chimica Acta, 304(1-2), pp.147–151. doi:https://doi.org/10.1016/s0009-8981(00)00412-5.
  • Wallace, A.M. (2000). Measurement of leptin and leptin binding in the human circulation. Annals of Clinical Biochemistry: International Journal of Laboratory Medicine, 37(3), pp.244–252. doi:https://doi.org/10.1258/0004563001899311.
  • Wild, D. (2013). The Immunoassay Handbook : Theory and Applications of Ligand binding, ELISA, and Related Techniques. 4th ed. Oxford ; Waltham, Ma: Elsevier.
  • Wilkinson, B. and Golabek, R. (n.d.). Assembly and method to improve vacuum retention in evacuated specimen containers. [online] Available at: https://patents.google.com/patent/US20090162587A1/en [Accessed 29 May 2024].
  • Wiseman, J.D. and National Committee For Clinical Laboratory Standards (1996). Evacuated tubes and additives for blood specimen collection : approved standard. 33rd ed. Wayne, Pa: Nccls.
  • Zahraini, H., Indrasari, Y.N. and Kahar, H. (2021). Comparison of K2 and K3 EDTA Anticoagulant on Complete Blood Count and Erythrocyte Sedimentation Rate. INDONESIAN JOURNAL OF CLINICAL PATHOLOGY AND MEDICAL LABORATORY, 28(1), pp.75–79. doi:https://doi.org/10.24293/ijcpml.v28i1.1735.
  • Zaninotto, M., Mion, M., Altinier, S., Forni, M. and Plebani, M. (2004). Quality specifications for biochemical markers of myocardial injury. Clinica Chimica Acta, 346(1), pp.65–72. doi:https://doi.org/10.1016/j.cccn.2004.02.035.
Human biospecimens