Anticoagulant

Commonly used anticoagulant in pathological laboratory

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Anticoagulants are substances that prevents blood coagulation. There are many chemical substances that prevent blood coagulation. All of them have some merits and demerits. There are variety of blood tests abd a single anticoagulant is not suitable for all the purpose. So, many types of anticoagulants are used in pathological laboratory.

EDTA

Full name is ethylene diamine tetra acetic acid. The other name is sequesterene.

EDTA blood
EDTA blood

Use

Fo3r routine haematological investigations.

Mechanism of action

This salts is a powerful chelating agents, binds with calcium. As calcium is a an important coagulating factors, coagulation cascade is hampered. Calcium is not available, so blood coagulation does not occurs.

Concentration

Need for edts salts is 1.5 ( +/- 0.25) mg/ ml of blood. At this concentration of salts, some shrinkage of cells occurs, so pcv decrease by 2-3%.

Disadvantages

If concentration is increased >2mg/ml, then edta cause shrinkage of rbc and wbc and there is degenerative changes. There is decrease of pcv, and increase of mchc.

Edta is unsuitable for coagulation study as it binds more strongly to other metal ions presents in blood. (see notes)

Colour code of EDTA vials

EDTA vial
EDTA vial

Violet generally.

Trisodium citrate

A 3.8% aqueous solution of of trisodium citrate is the anticoagulant of choice for coagulation study and estimation of esr.

Mechanism of action:

It binds with calcium and chelates calcium. Calcium is not available, so blood coagulation does not occurs.

Use

  1. Estimation of esr- 4 volume of venous blood is diluted with1 volume of 3,8% citrated solution (4:1 ratio).
  2. Prothrombin time : 9 volume of blood are added to 1 volume of sodium citrate in the ratio of 9:1.

Oxalates

Potassium, sodium and ammonium oxalates act as chelating agents. They interact with calcium and binds with calcium- resulting formation of calcium oxalates.

Use

Potassium and sodium oxalates are used in the concentration of 2 mg/ml of blood.

Double oxalate

Two oxalates are mixed with the ration 3 parts of ammonium oxalates and 2 parts of of potassium oxalates.

The mixture is used in the concentration of 2mg/ml of blood.

Preparation of double oxalates

1.2 mg ammonium oxalates are taken in a vial and mixed with 0.8 mg of potassium oxalates and 100 ml distilled water.

This solution contains 20 mg of oxalates per ml of mixture. 0.1 ml of this mixture is pipette in a collection container and dried in an incubator or oven.

This  is sufficient for 5ml of blood.

Uses

  1. Haemoglobin estimation
  2. Haematocrit value
  3. Determination of specific gravity of whole blood or plasma.

Disadvantages

Double oxalate cause morphological changes of rbc and wbc, so smears are not suitable for morphological study of cells.

This anticoagulant is not commonly used now a days.

Sodium fluoride

Sodium fluoride is a commonly used anticoagulant for the estimation of blood sugar.

Mechanism of action

  1. It forms calcium fluoride with calcium and chelates calcium from blood therefore act as a anticoagulants.
  2. Moreover, it block the enzyme phosphorylase in the glycolytic pathway in the red blood cells. Therefore no glycolysis occurs and glucose value does not alter in blood. When estimation is delayed, thymol is used as preservatives along with sodium fluoride.

Heparin

Heparin is another commonly used anticoagulant for some special tests.

Mechanism of action

  1. Heparin in powder or liquid forms act by inhibiting thromblplastin formation in coagulation cascade.
  2. It also act as an antithrombin by inhibiting the action of thrombin to fibrinogen.

Uses

  • It is used in the concentration  of 10-20iu ( 0.1-0.2 mg) /ml of blood
  • It does not alter the size of rbc and therefore it it used for osmotic fragility tests
  • It is used gor g6pd deficiency and pk deficiency study.
  • Used for chemical estimation of plasma  iron and other substances.
  • Clinical use for the treatment of deep vein thrombosis, pulmonary embolism and heart surgery.

Disadvantages

  • It is expensive
  • Heparinised blood is unsatisfactory for blood films and counts, because it cause wbc and platelets to clump.
  • It is not suitable for general use.

Note

The international council for standardization in haematology recommended the westergren method as the method of choice. The westergren method uses citrate, a liquid-based anticoagulant, resulting in dilution of blood, inaccuracies of which significantly affect the esr. Modifications of the reference method, also called the modified westergren method, are also widely used and use edta in place of citrate as an anticoagulant. Edta is a solid-based anticoagulant that does not cause significant dilution (<1%), reducing the errors of dilution ( More Information)

For esr tube percentage of sodium citrate is 3.8%  when we are doing esr with glass tubes and with plastic esr  tubes, the tubes contain 3.2% sodium citrate solution as anticoagulant. And 3.2% citrate is also recommended and  be used for all coagulation tests (pt ppt, etc) to achieve accurate  results . (more information)

Edta binds calcium ions more strongly than citrate, edta also binds structural calcium in the proteins, partly inactivating them. Besides calcium, edta also binds other divalent metal ions like copper quite strongly. Factor viii and factor v are copper-binding proteins (similarity with ceruloplasmin) and dissociate. [ More information]

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