RELEVANCE OF ANTICOAGULANT

ANTICOAGULANTS AND THEIR EFFECT  ON BLOOD

Introduction

An anticoagulant is a substance that prevents blood from clotting. Anticoagulants can be used in vivo

(1) As medication eg in thrombotic disorders such as deep vein thrombosis, pulmonary embolism, myocardial infarction and strokes.

(2) In medical equipments such as test tubes, blood transfusion bags and renal dialysis equipment.

Anticoagulants achieve their effect by suppressing the synthesis or function of various clotting factors that are normally present in the blood. Anticoagulants are  generally of two types.

 One type is heparin, which is a mixture of muco-polysaccharides that promote the activity of antithrombin III, a blood plasma protein that inactivates thrombin (an e nzyme that promotes clotting).

The other type consists of the coumarin derivatives, which interfere in the metabolism of vitamin K in the liver. Since vitamin K is an important element in the synthesis of various clotting factors, interference in the metabolism of that vitamin gives rise to clotting factors that are defective and hence inactive in the blood. Coumarin derivatives differ from heparin primarily in their longer duration  of action; of these, the most widely used is warfarin. A major side effect of anticoagulant therapy is hemorrhage. Anticoagulants are also used in drawing and storing blood.

EDTA (ethylene diamine tetraacetic acid)

 EDTA (ethylenediaminetetraacetic acid) is the most commonly used anticoagulant in evacuated tubes. It inhibits the clotting process by removing calcium from the blood. EDTA is able to form a complex with metal ions. 

 EDTA is synthesized on an industrial scale from ethylenediamine, formaldehyde,  and a source of cyanide (HCN or NaCN)

This chemical has been used to prevent clotting in blood

specimens since the early 1950s and has certain advantages over other anticoagulants. EDTA’s most distinct characteristic is that it does not distort blood cells, making it ideal for hematology use.

 Enough EDTA must be present to prevent coagulation, but excessive amounts cause morphological changes in blood cells. For instance, K₂EDTA is present in a concentration of 1.5 to 2.0 mg/ml of blood, it does not have any significant effect on the blood count parameters. All tubes should be inverted several times (8-10) to ensure thorough mixing and, therefore, proper anticoagulation.

  Effect of EDTA on Red Blood Cells (RBCs):

High quality blood smears can be made from the EDTA tube as long as they are made within 2-3 hours of the blood draw.

Ø Smears made from EDTA tubes that sit at room temperature for more than 5 hours often have unacceptable artifact of the blood cells (echinocytic RBCs, spherocytes, and necrobiotic leukocytes).

Ø If a tube is not filled to its full volume of draw, the additive to blood specimen ratio is affected, resulting in too high a concentration of EDTA.

 In high concentrations, EDTA causes red cells to shrink because of hyper-tonicity of the plasma with increased ionic concentration and may create artifacts that make RBC morphology difficult to interpret. An excess of EDTA affects both erythrocytes and leukocytes, causing membrane damage.

Effect of EDTA on Platelets:

EDTA reduces platelet activation by protecting the platelets during contact with the glass tube that may initiate platelet activation. Activation causes platelets to clump in the presence of calcium and platelets adhere to the glass surface at a rapid rate. Chelation of calcium using EDTA results in decreased platelet adhesion or retention to glass.

Ø Pseudothrombocytopenia can complicate an accurate determination of a platelet count in a patient with an underlying thrombocytopenic disorder.  Platelet clumping may be a result of poor mixing - too little and/or too late,  and/or a small, whole blood clot or very small fibrin clots in the EDTA-anticoagulated specimen.

Ø Additionally, the improper collection of the blood sample may cause thrombin release and a falsely low platelet count due to platelet aggregation.

Ø Clotting can also be the result of insufficient EDTA, usually caused by overfilling the vacuum tube, or poor solubility of EDTA (most commonly Na₂ EDTA).

It is important to be able to distinguish between reduced platelet counts due to technique related variables or due to a patient’s medical condition. There are two patient conditions in which the presence of EDTA causes the platelets to clump.

1.     The first patient condition, platelet satellitism, is observed on a differential smear as a “halo” or ring of platelets surrounding white blood cells (WBCs). If the specimen is collected in an anticoagulant other than EDTA, this ring does not occur. Antibodies in most cases probably cause this in vitro phenomenon.

2.     The second patient condition is pseudothrombocytopenia due to EDTA. This occurs in certain patients who have antibodies that can bind to platelets. When EDTA is added to the blood, the antibodies are activated and cause platelet clumping. This phenomenon is sometimes temperature dependent, with more platelets clumping as the blood cools to room temperature after collection. Again, if the specimen is collected in an anticoagulant other than EDTA, this does not occur.

One study showed that when the blood sample was incubated at 37°C, platelet clumping was not seen, and an accurate count could be obtained.

EDTA prevents platelets from clumping on the slide, making it easier to more accurately estimate platelet counts. When a test result shows a low platelet count, a differential smear should be made to help determine if the cause of the low platelet count is due to a patient condition or mixing of blood with EDTA.

Effects of EDTA on Leukocytes (WBCs):

Studies have demonstrated that the WBC count remained stable for at least 3 days when EDTA anticoagulated blood was stored at room temperature.

Neutrophils and monocytes appeared to be the cells most sensitive to storage in EDTA, whereas lymphocytes were the most stable. With respect to the WBC morphologic characteristics of EDTA-anticoagulated blood on storage at ambient (20-24°C) temperatures, a slight vacuolization of monocytes was found after one hour, progressing to moderate after four hours; a slight vacuolization of neutrophils was found after 3-4 hours, progressing to moderate after 6 hours. Only minimal changes in the WBC morphologic characteristics have been reported on storage at 4°C for as long as 12 hours.

EDTA Choices:

There are three different forms of EDTA. EDTA is available in disodium (Na₂EDTA), di-potassium (K₂EDTA) and tri-potassium (K₃EDTA) salts. K₂EDTA and Na₂EDTA salts are commonly used in dry form; K₃EDTA is normally used in liquid.

K₃EDTA is dispensed as a liquid and thus causes a slight dilution of the specimen. This salt also has been shown to affect the red blood cell size more at increased concentrations and on storage than the K₂EDTA.

Therefore, K₂EDTA is recommended as the anticoagulant of choice in specimen collection for blood cell counting and sizing. K₂EDTA is spray-dried on the wall of the tube and will not dilute the sample and is recommended by ICSH (International Council for Standardization in Haematology) and CLSI (Clinical and Laboratory Standards Institute) for haematology testing.

Tri-sodium citrate (Na₃C₆H5O₇)

Trisodium citrate was first used as an anticoagulant in blood transfusions in 1914, by Albert Hustin, a Belgian physician and Luis Agote, an Argentine physician. It continues to be used today in blood collection tubes and for the preservation of blood in blood banks.

The citrate ion chelates calcium ions in the blood by forming calcium citrate complexes, disrupting the blood clotting mechanism.

Sodium citrate is effective as an anticoagulant due to its mild calcium-chelating properties.

Sodium citrate is not used as the routine anticoagulant for haematological tests because it has a standard blood dilution of 1 part of anticoagulant to 9 parts blood. This means haematology analyser would have to be re-calibrated to prevent giving erroneous results.

When sodium citrate is used for erythrocytes sedimentation rate (ESR) it is used in a fixed ratio of 1part of the anticoagulant to 4parts of blood. It is thought that a hypothetical factor called sedimentin that play a role in ESR process, is rather stable in this anticoagulant.

The frequently used 3.8% (0.129 mmol/L) citrate is somewhat hyperosmolar for red cells, resulting in shrinkage of red cells and artifactual lowering of the haematocrit.

The preferred 3.2% (0.109 mmol/L) citrate solution is iso-osmolar and hence should not affect the haematocrit determination.

Sodium citrate is the anticoagulant of choice for blood coagulation studies because, the clotting factors assessed are very stable in sodium citrate but disintegrate rapidly in EDTA because of it highly  potent calcium chelation effect

Unfortunately, other anticoagulant such as heparin, inhibit multiple clotting factors and so cannot be used.

Heparin

Heparin anticoagulant prevents blood clotting by promoting the activity of anti-thrombin III, a blood plasma protein that inactivates thrombin (an enzyme that promotes clotting).

Heparin is an anticoagulant used in clinical chemistry and special chemistry testing. It is the recommended anticoagulant for many determinations using whole blood or plasma specimens because of its minimal chelating properties, minimal effects on water shifts, and relatively low cation concentration.

Heparin is the only anticoagulant that should be used in a blood collection device for the determination of blood pH, blood gases, electrolytes and ionized calcium, lithium,  zinc and carboxyhaemoglobin .

There are currently three salts of heparin that are commonly used in blood collection devices: ammonium, lithium and sodium.

Lithium heparin is the recommended form of heparin to be used because it is least likely to interfere when performing tests for other ions. Lithium heparin is essentially free of extraneous ions. It should not be used for collection of blood for lithium levels.

Heparin should not be used for coagulation or haematology testing. Heparin is not recommend ded because of frequently developing platelet clumps that interfere with the morphological interpretation of platelets and platelet counts. Heparin is also not good for blood films because it forms a purple/blue hue on stained films.

Acid Citrate Dextrose and citrate phosphate dextrose (CPD)

Acid citrate dextrose (ACD) is an important blood anticoagulant and preservative. This anticoagulant is mainly used for the blood bank studies, HLA phenotyping, flow cytometry testing, tissue typing, DNA testing. At present, there are two widely used forms of acid citrate dextrose: namely solution A and solution B.

Solution A comprises of 22.0 g/l Trisodium citrate, 8.0 g/l citric acid, 24.5 g/l dextrose. Solution B comprises of 13.2 g/l trisodium citrate, 4.8 g/l citric acid, 14.7 g/l dextrose.

For the purpose of anticoagulant for further laboratory studies, either solution A or solution B is acceptable. However, for the purpose of blood preservative, solution A is preferred.

ACD is works by weakly chelating calcium ions thus preventing blood clotting. The dextrose and citric acid make ACD a good red blood cell preservative.

However, chelation of calcium upon storage causes hypocalcaemia in the blood and when given for transfusion results in symptoms such as numbness in fingers, nausea, etc.

Because of this, ACD is now mainly replaced by citrate phosphate dextrose with adenine (CPDA). The storage life-span of RBCs in ACD is 21days whiles in CPDA, red cells could be stored for 35days.

CPDA-1 contains 26.3g trisodium citrate, 3.27g citric acid, 2.22g sodium dihydrogen phosphate, 31.8g dextrose and 0.275g adenine per liter. 14ml is used for 100ml of blood.

CPDA-2 the amount of dextrose is increased to 44.6g and that of adenine increased to 0.55g. the additional, adenine hasten the loss of 2,3 DPG from RBC a substance from within RBCs during glycosis that has more affinity for Hb thus it up production facilitating release of oxygen from Hb in the tissues.

Oxalates

Oxalates of ammonium and potassium was extensively used in haematology an anticoagulants however, these are no more in use with the advent of EDTA.