Types of Anticoagulant, Additives and Their Adverse Effects

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Evacuated blood collection tubes without anticoagulant are used to produce serum (or as a discard tube), whereas other types of evacuated tubes may contain an anticoagulant or additive. The additives range from those that promote faster blood clotting to those that preserve or stabilize specific analytes (such as glucose) or cells. The use of additives at the proper concentration in evacuated tubes improves the accuracy and consistency of test results while also allowing for faster turnaround times in the laboratory.


Inorganic additives, biochemical additives, and gel in blood collection tubes come in a variety of forms. Tripotassium ethylenediaminetetraacetate (K3 EDTA), dipotassium ethylenediaminetetraacetate (K2 EDTA), sodium citrate, and heparin are examples of commonly used anticoagulants. Each type of anticoagulant prevents whole blood coagulation. To avoid the introduction of errors into test results, the proper proportion of anticoagulant to whole blood must be used. The laboratory procedure manual should specify the specific type of anticoagulant required for a procedure.


The salts of the chelating (calcium-binding) agent EDTA are recommended by the International Council for Standardization in Haematology (ICSH) and CLSI as the anticoagulant of choice for blood cell counting and sizing because EDTA causes less shrinkage of RBCs and less increase in cell volume on standing. The interior surface of evacuated plastic tubes is spray-dried with EDTA. The correct EDTA-to-whole-blood ratio is critical because some test results will be affected if the ratio is not correct. Excessive EDTA causes erythrocyte shrinkage, which affects tests like the manually performed packed cell volume (microhematocrit).

K2 EDTA and K3 EDTA are found in evacuated tubes. This anticoagulant works by chelating ionized calcium (Ca2+). This process produces an insoluble calcium salt, which prevents blood coagulation.

In hematology, EDTA is the most commonly used anticoagulant for the complete blood cell count (CBC) or any of its component tests: hemoglobin, packed cell volume, total leukocyte count and leukocyte differential count, and platelet count. The anticoagulant of choice in the modified Westergren erythrocyte sedimentation rate (ESR) method is EDTA. Furthermore, EDTA is routinely used in blood banking testing such as blood grouping, Rh typing, and antibody screening.

In molecular diagnosis, K2 EDTA with gel is used to test plasma. K3 EDTA can be used to test for viral markers.

Sodium Citrate

There are sodium citrate solutions in concentrations of 3.2 percent and 3.8 percent. Sodium citrate removes calcium from the coagulation system by precipitating it into an unusable form. Because of its mild calcium-chelating properties, sodium citrate works well as an anticoagulant. Sodium citrate is used as an anticoagulant in activated partial thromboplastin time (APTT) and prothrombin time (PT) testing, as well as the classic Westergren ESR. In blood collection tubes, the correct anticoagulant-to-whole-blood ratio of one part anticoagulant to nine parts whole blood is critical. An excess of anticoagulant can alter the expected dilution of blood and cause errors in the results. Because of the dilution of anticoagulant in blood, sodium citrate is generally unsuitable for most other hematology tests.


Heparin is used as an anticoagulant both in vitro and in vivo. Heparin works as an antithrombin, or a substance that inhibits the clotting factors thrombin and factor Xa. This thrombin inactivation is caused by heparin complexing with the antithrombin III molecule and catalyzing the inhibition of thrombin.

Because it is less likely to interfere with other ion tests, lithium heparin is the preferred form of heparin for use in laboratory testing. The in vitro formation of fibrin in heparinized plasma opposes heparin’s anticoagulant action and can result in the formation of fibrin in the plasma. Several specimen-processing steps are recommended to help ensure a high-quality heparinized plasma sample and aid in the reduction of latent fibrin formation.

Heparin is the only anticoagulant that should be used to measure pH, blood gases, electrolytes, and ionized calcium. Heparin is used to coat “micro” (capillary blood) collection tubes before they are used in hematology or clinical chemistry procedures.

Heparin is an ineffective anticoagulant for many hematologic tests, including Wright-stained blood smears, because it causes the smear to stain too blue.

Types of Additives


Thrombin, an enzyme that converts fibrinogen to fibrin, is one type of additive. Because of their quick clotting time, thrombin tubes are frequently used for “stat” serum testing.

Sodium Fluoride

Sodium fluoride (NaFl), a dry additive and weak anticoagulant, is primarily used to preserve blood glucose specimens to prevent glycolysis or glucose destruction.


The additive gel’s function is to create a physical and chemical barrier between the serum or plasma and the cells. Their use provides significant advantages in the collection, processing, and storage of specimens in primary tubes.

Because of the way they respond to applied forces, separator gels can provide barrier properties. When blood is drawn into the evacuated gel tube and centrifugation begins, the g-force applied to the gel reduces its viscosity, allowing it to move or flow. Thixotropic materials are those that have these flow characteristics. When the centrifuge is turned off, the gel becomes an immobile barrier between the supernatant and the cells. Because of the composite nature of gels, these gel tubes have an infinite shelf life.

Anticoagulants and Additives tubes colors

LavenderK2 EDTA (spray-coated plastic tube) K3 EDTA (liquid in glass tube)
PinkK2 EDTA (spray-coated plastic tube)
Light green/marbled greenSodium heparin, lithium heparin, ammonium heparin
Light blue or clear (Hemogard closure)Buffered sodium citrate, CTAD
White†K2 EDTA with gel
Red/light gray‡ or clear (Hemogard closure)None (plastic)
RedSilicone coated (glass) Clot activator, silicone coated (plastic)
Gold/marbled redClot activators and gel
GraySodium fluoride
YellowSPS: blood culture (Hemogard closure) Acid citrate dextrose: HLA studies (color stopper)
Royal blueNo additives for toxicology, trace metals (Hemogard)
Orange/marbled yellowThrombin
BlackSodium citrate
TanSodium heparin
Examples of BD Stopper Colors for Venous Blood Collection
No Additive TubesNoneWhiteBlack
Coagulation Tubes3.2% Sodium citrateLight blueBlack
Serum TubesClot activator with gelRed or goldYellow or gold
Heparin TubesLithium heparinGreenBlack
 Lithium heparin with gelGreenYellow
 Sodium heparinGreenGreen
EDTA TubesK2 EDTALavenderBlack
 K2 EDTA with gelLavender or whiteYellow
 K3 EDTALavenderBlack
Glycolytic Inhibitor TubesNaFl/KOGrayBlack
Trace Element TubesSodium heparinRoyal blueBlack
Examples of VACUETTE® SAFETY Cap Color Codes for Venous Blood Collection

Adverse Effects of Additives

The additives chosen for specific determinations must not alter the blood components or interfere with the laboratory tests to be performed. The following are some of the negative consequences of using an incorrect additive or the incorrect amount of additive:

  • Interfere with the test. The additive may contain a substance that is the same as, or reacts in the same way as, the substance being measured; for example, sodium oxalate may be used as an anticoagulant in a sodium determination.
  • Removal of constituents. For example, using an oxalate anticoagulant for a calcium determination may remove the constituent to be measured; oxalate removes calcium from the blood by forming an insoluble salt, calcium oxalate.
  • Effect on enzyme action. The additive may have an effect on enzyme reactions, such as when NaFl is used as an anticoagulant in an enzyme determination; NaFl destroys many enzymes.
  • Alteration of cellular constituents. The use of an older anticoagulant additive, oxalate, in hematology, for example, can alter cellular constituents. When oxalate is used as an anticoagulant, the cell morphology changes; RBCs become crenated (shrunken), vacuoles appear in granulocytes, and bizarre forms of lymphocytes and monocytes appear quickly. Another example is the use of heparin as a blood anticoagulant in the preparation of blood films to be stained with Wright’s stain. Heparin produces a blue background with Wright’s stain unless the films are stained within 2 hours.
  • Incorrect amount of anticoagulant. If too little additive is used, there will be partial clotting of whole blood. This causes problems with cell counts. In comparison, using too much liquid anticoagulant dilutes the blood sample, interfering with certain quantitative measurements.

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About the Author: Labweeks

KEUMENI DEFFE Arthur luciano is a medical laboratory technologist, community health advocate and currently a master student in tropical medicine and infectious disease.


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