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