Anticagulants

Most tests performed in the hematology laboratory involve anticoagulated blood. Once the blood has left the body, a series of reactions occurs causing blood to clot within minutes. To prevent coagulation from occurring, a substance called an anticoagulant is mixed with the blood. Three anticoagulants are used in the hematology laboratory:-

1. Ethylenediaminetetraacetic acid (EDTA)
2. Sodium citrate.
3. Heparin. 

EDTA:-

EDTA is the most commonly used anticoagulant in the routine hematology laboratory. Sample collection tubes can contain one of three different salt forms of EDTA:

1. Disodium (Na₂EDTA).
2. Dipotassium (K₂EDTA).
3. Tripotassium (K₃EDTA).

The sample collection tube’s stopper is color coded in lavender to indicate the presence of EDTA. EDTA prevents coagulation by chelating calcium, anecessary component of the coagulation cascade. Its removal inhibits the coagulation process. The optimal concentration is 1.5 mg of EDTA per mL blood. Tests using EDTA samples include:

• Complete blood count (CBC)
• Hematocrit
• Peripheral blood smear examination
• Platelet count,
• Reticulocyte count,
• Flow cytometry. 

Ideally, the sample should be used within 6 hours of collection for the majority of these tests. The sample’s stability can be extended to 24 hours with storage at 4°C for certain tests such as the CBC and platelet count. If peripheral blood smears are to be prepared, they should be made within 3 hours of collection. After 3 hours at room temperature, degenerative changes can be observed by examining a Wright-stained blood smear. These changes include:

• Leukocytes with vacuoles.
• Irregular cytoplasmic borders.
• Irregularly shaped nuclei.
• Platelets increase in size. 

Excess anticoagulant causes erythrocyte shrinkage. Concentrations of more than 2 mg of EDTA per mL blood cause false decreases in the microhematocrit and erythrocyte sedimentation rate (ESR).

Sodium Citrate:-

Sodium citrate is the recommended anticoagulant for coagulation studies. The Clinical and Laboratory Standards Institute (CLSI) recommends the use of 3.2% sodium citrate. The sample collection tube’s stopper is color coded in light blue to indicate the presence of sodium citrate. Sodium citrate prevents coagulation by binding calcium in a soluble complex. The appropriate ratio of anticoagulant: blood is 1:9 for coagulation studies and 1:4 for erythrocyte sedimentation rate (ESR) test.

Heparin:-

Lithium heparin is the CLSI recommended salt of heparin to be used for laboratory testing. The sample collection tube’s stopper is color coded in green to indicate the presence of heparin. Heparin’s interaction with antithrombin prevents coagulation. The interaction leads to the inhibition of thrombin. The recommended concentration for sample collection tubes is 15–30 units heparin/mL blood. Lithium heparin is specifically recommended for the following laboratory tests:

• Ammonia
• Carboxyhemoglobin
• Blood gases
• Zinc
• Potassium.
• Osmotic fragility test.

In hematology, lithium heparin is the appropriate anticoagulant for the osmotic fragility test. The use of heparin for routine hematology procedures is not appropriate. Heparin can affect the platelets and leukocytes, causing them to clump. In addition, heparin causes morphologic distortion of platelets and leukocytes and tends to cause a bluish discoloration of the background of blood films stained with a Romanowsky stain such as Wright stain.