Anticoagulants Used in Hematology Laboratory

 

Top 5 Anticoagulants Used in Hematology Laboratory | Biology

The principal uses of anticoagulants are in vitro to prevent clotting of blood for trans­fusion or diagnostic use and in vivo to prevent development and enlargement of thrombi.

The following points highlight the top five anticoagulants that are commonly used in hematology. The anticoagulants are: 1. Double Oxalate 2. Ethylene Di-Amine Tetra Acetic Acid 3. Heparin 4. Sodium Citrate 5.  Sodium Fluoride.

Anticoagulant # 1. Double Oxalate:

0.5 anticoagulant for 5 ml of blood.

Chemical Action:

This anticoagulant removes the free calcium ion from solution through the addition of ammonium and potassium oxalate. Calcium is precipitated as insoluble calcium oxalate.

Preparation:

i. 1-2 gram ammonium oxalate and 0.8gm potassium oxalate are dissolved in 100ml of distilled water.

ii. 0.5ml of this solution is added to each of a series of tubes and evaporated to dryness at 37°C higher temperature, decompose the exalted.

Use:

Blood taken into this anticoagulant is unsuitable for morphological examination the red cells commerce to crenate and the white exhibit bizarre nuclear patterns.

Anticoagulant # 2. Ethylene Di-Amine Tetra Acetic Acid (EDTA):

EDTA can be found in three salt forms:

1. Tri-Potassium EDTA

2. Di-Sodium EDTA

3. Di-Lithium EDTA

Also, EDTA can be crystalline or liquid. Liquid EDTA tubes, requires specific filling volume to avoid dilution effect. So, blood: anticoagulant ratio must be maintained (this is applicable to all anticoagulants). EDTA is also known as Versene or Sequestrene.

EDTA acts by chelating/removing ionized calcium (calcium is required for blood to clot, so when it is removed blood will not clot). Generally tri-Potassium EDTA is better than di-Sodium EDTA and di-Lithium EDTA.

Always, be sure to mix blood with anticoagulant in a manner that guarantee proper complete mixing, by gentle repeated inversion of the tube, inversion for at least 20 times, do not shake or use vigorous inversion, since this may cause hemolysis, and disintegration of cells, and the final effect will be erroneous low results for cellular components of blood EDTA is the most commonly used anticoagulant in the hematology laboratory, and is the anticoagulant of choice for the CBC.

Excess EDTA (i.e. more EDTA, you fill less blood volume, so EDTA is in excess), causes shrinkage of RBC’s, causing falsely/erroneously reduced hematocrit (HCT), and subsequent increase in MCHC and decrease in MCV (MCV and MCHC are RBC indices that will be studied later).

Platelets are also affected, they will swell and subsequently disintegrate, causing erroneously high platelet count, since platelets will be disintegrated into more than one fragment, and each fragment will be counted as one platelet

Anticoagulant # 3. Heparin:

Heparin is an acid mucopolysaccharide, it acts by complexing with antithrombin to prevent blood clotting (antithrombin is one of the natural/physiological inhibitors of blood coagulation). It is not suitable for blood films staining, since it gives too blue coloration to the background, when films are stained with Romanovsky stains, also, heparin may cause leukocyte and platelet clumping, this is why heparin is not suitable for routine hematology tests.

It is the preferred anticoagulant for osmotic fragility test. Heparin also is used in capillary tubes for spun hematocrit (HCT) (heparin cover the entire capillary tube glass), these capillary tubes are also called microhematocrit capillary tubes. Heparin is also used for L.E. cell preparation (L.E. = Lupus Erythromatosus).

i. Heparin is found in basophil and mast cell granules

ii. Heparin is used therapeutically as an in vivo anticoagulant.

Anticoagulants Commonly Used in the Hematology Laboratory and their Use:

No. Anticoagulant Hematology Laboratory Use Universal Color Code

1. EDTA Routine Hematology Procedures. Lavender, Pink

2. Sodium citrate Coagulation, Platelets Tests, ESR. Blue

3. Heparin Osmotic Fragility, Spun Hematocrit Green, Brown

Preparation:

a. 0.4gm of prepared heparin is dissolved in 100ml of distilled water.

b. 0.25ml of this solution is added to each of a series tubes and evaporated to dryness of 37°C.

Anticoagulant # 4. Sodium Citrate:

This is most commonly used anticoagulant, mainly used to prepare citrated plasma for the evaluation of coagulation disorders as it is not inhibitory to calcium ions.

Is the anticoagulant of choice for coagulation and platelet function tests, also is used for ESR (erythrocyte sedimentation rate test). It acts by precipitating calcium, thus it will not be available for clotting process.

It came in a liquid form, as 3.8% tri-sodium citrate. For coagulation testing, the ratio of 9 volumes of blood to one volume of anticoagulant (9 volumes blood: 1 volume anticoagulant) is very critical (very important), as variation from this ratio may cause errors. For ESR (4) volumes of blood to one volume of anticoagulant is used (4: 1).

Generally, this anticoagulant is not suitable for routine hematology testing. From this we conclude that sodium citrate acts as anticoagulant and as diluent (as in the case of ESR). Because of its dilution effect it can’t be used for CBC.

Preparation:

3.8g Trisodium citrate is dissolved in 100ml of distilled water

Anticoagulant # 5. Sodium Fluoride:

This anti-coagulant is used for preparing blood specimens for the determination of glucose and urea in plasma by non-enzymatic methods. Fluroide inhibits glycolic enzymes and thereby prevents loss of glucose during transportation or delay in specimen handling. As fluoride is not a strong anticoagulant, it is mixed with oxalate.

In Vitro Anticoagulants:

Essentially two categories of chemicals are used: those employed as anticoagu­lants in samples of blood intended for physical or chemical examination and those employed to preserve blood for transfusion. Certain of these agents can be used to prevent clotting both in vitro and in vivo (heparin), while others may be good for use in vitro, but are not practical in vivo because of their toxicity (oxalates).

(A) Anticoagulant agents used in laboratory examination of blood include:

(i) Sodium oxalate (20%) → 0.01 ml/ml of blood.

(2 mg/ml of blood)

(ii) Sodium citrate (25%) → 0.01 ml/ml of blood (2.5 mg/ml blood)

(iii) Edetate disodium (Sod. versenate) → (EDTA) (2%) → 0.01 ml/ml blood (0.2 mg/ml blood)

(iv) Heparin sodium – 75 i.u./10 ml of blood (1 mg = 100 i.u)

Any of these anticoagulants may be added to sample tubes in the desired amounts and evaporated to dryness. Sterile vacuum tubes that contain appropriate anticoagu­lants and double needles for ease in blood collection are now available.

(B) Anticoagulants used for blood and blood plasma transfusion include:

(C) Other means also may prevent or retard coagulation.

(i) Cold at 2-5°C.

(ii) Collection of blood into a receptable having smooth and unwettable walls, e.g. paraffin, or silicone coated.

Systemic Anticoagulants:

Two types of anticoagulants are used therapeutically for preventing enlargement of thrombi.

(i) Heparin-used parenterally. It has a direct and almost instantaneous action on the coagulation process.

(ii) Coumarin derivatives-used orally. They have an indirect anticoagulant effect by acting as vitamin K antagonists.

Therefore, action by the coumarin derivatives is delayed for several hours. For emergency treatment, heparin is used first then may be followed by the coumarin derivatives.

Other anticoagulants include edentate sodium, sod. oxalate, and sod. citrate, which hinder coagulation by combining c̅ Ca2+. That’s why these are not useful in vivo. Both categories of the systemic anticoagulants act by inhibition of the action of formation of one or more clotting factors.

Heparin:

(i) Heparin sodium is the only anticoagulant drug used parenterally. Heparin is present in mast cells along with histamine and serotonin and prepared commercially from bovine lung and porcine intestinal mucosa.

(ii) Heparin is highly negatively charged mucopolysaccharide, having larger molecular size. That’s why it is administered parenterally.

Mech. of Action and Pharmacological Effects:

(i) Heparin prolongs clotting time to blood both in vivo and in vitro.

(ii) Heparin prevents fibrin formation in the process of coagulation.

(a) It increases the activity of antithrombin III.

(b) Antithrombin III then inhibits the conversion of prothrombin to thrombin by thromboplastin.

(c) Antithrombin III also directly inactivates thrombin (only in presence of heparin).

[Heparin exerts its anticoagulant action by combining with the endogenous α – 2 globulin called antithrombin III or heparin cofactor’. This combination markedly accelerates the antagonistic action of antithrombin III on thrombin. With thrombin inactivated, conversion of fibrinogen to fibrin is restricted. The heparin antithrombin complex is also inhibitory to the activated forms of other clotting factors-factors IX, X, XI, XII and kallikrein.]

(iii) Heparin decreases aldosterone secretion, increases the concentration of free thyroxine.

(iv) It slows wound healing and probably also depresses cell-mediated immunity.

Therapeutic Uses:

(i) In prophylaxis and treatment of venous thrombosis and pulmonary embolism.

Venous (red) thrombi consist of a fibrin network en-mashed with RBCs and platelets. The anticoagulants are generally ineffective in the treatment of arterial (white) thrombi, made up of adhering platelets. Arterial thrombi are treated with the antithrombotics and thrombolytics.

(ii) In atrial fibrillation with embolization.

(iii) In diagnosis and treatment of chronic consumptive coagulopathies.

(iv) As an anticoagulant in blood transfusion.

(v) For prevention of clotting in arterial and heart surgery.

(vi) For prevention of cerebral thrombosis.

Heparin sodium for inj. contains 10mg (or 1000 units) or more/ml. For i.v. admn., dilution is made with normal saline or 5% glucose (10 mg per 1000 ml) and is dripped slowly as needed.

Coumarin Derivatives:

(i) Drugs under this group are Dicumarol (Bishydroxy-coumarin) and warfarin [3-(α-acetonyl- benzyl) -4 hydroxycoumarin]. Coumarin, normally present in some species of sweet clover, has no anticoagulant action.

(ii) Warfarin sodium is the drug of choice and is considered the prototype of the coumarin- derived anticoagulants.

(iii) These are oral anticoagulants.

(iv) These are vitamin K antagonists.

Mechanism of Action and Pharmacological Effects:

(i) The oral anticoagulants are antagonists of vit. K. Coagulation factors II (prothrombin), VII, IX and X and anticoagulant protein is ‘C’ and ‘S’ are biologically inactive unless certain glutamic acid residues (9 to 12 in number) are carboxylated by a microsomal enzyme system that utilizes reduced vitamin K as a cofactor. Oral anticoagulants prevent reduction of vitamin K.

So, coumarin-derived anticoagulants interfere with vit-K- dependent synthe­sis of ‘active’ coagulation factors II, VII, IX, and X and proteins ‘C’ and ‘S’.

(ii) The oral anticoagulants are effective only in vivo, and take 8-12 hours for action.

Therapeutic Uses:

(i) Widely used in secondary prophylactic treatment of venous thrombosis and Pulmonary embolism to prevent the recurrence or extension of venous thrombus formation.

(ii) Since oral anticoagulants have no effect on platelet, they are not used in the treatment of thrombotic disease in the arterial system.

Drug Interactions with Oral Anticoagulants:

(i) Acetylsalicylic acid, cimetidine, phenylbutazone, oxyphenbutazone, cotrimoxazole, metronidazole, disulfiram, clofibrate, etc. increase the re­sponse to anticoagulants.

(ii) Rifampin, cholestyramine, glutethimide and barbiturates reduce the effi­cacy of anticoagulants.

Coagulants:

Classification:

1. Fresh whole blood or plasma.

2. Vitamin K:

(i) K1 (from plants, fat soluble) → Phytonadione (Phylloquinone)

(ii) K2 (produced by bacteria) → Menaquinones.

(iii) K3 (Synthetic):

(a) Fat soluble → (i) Menadione (ii) Acetomenaphthone

(b) Water soluble → (i) Menadione Sod. bisulfite.

(ii) Menadiol Sod. diphosphate.

3. Miscellaneous:

(i) Fibrinogen (human)

(ii) Antihaemophilic factor

(iii) Tissue extract

(iv) Adrenochrome monosemicarbazone

(v) Rutin

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