ANTICOAGULANTS
An anticoagulant is a chemical or substance that stops or slows the clotting of blood. These
anticoagulants can be made from salts of citric acid or tetra-acetic acid and oxalic acids.
Note: Haematological tests are performed on whole blood which is anticoagulated or capillary
blood before it clots. Examples of haematological tests that are performed on whole
anticoagulated blood are:-WBC total count, Platelet total counts, Haemoglobin estimation test
, Reticulocyte count test, thin blood film comment, Etc.
There are mainly four types of anticoagulants used in the haematology laboratory namely:-
Ethylene diamine tetra acetic acid (EDTA)
Trisodium citrate anticoagulant
Heparine anticoagulant
Oxalate anticoagulant
Mode of action of anticoagulants
EDTA and trisodium citrate anticoagulants acts by chelating the calcium ions. Oxalates acts by
precipitating the calcium ions. Heparine works by neutralizing thrombin in presence of the
antithrombin iii.
Note : For long term storage of the red blood cells i.e. for transfusion purposes trisodium citrate
anticoagulant is preferred in combination with other components. Examples of anticoagulants
used in the blood transfusion laboratory:-
ACD
CPDA
CPD
CP2D
EDTA (ETHYLENE DI-AMINE TETRA-ACETIC ACID)
This is also called sequestrene anticoagulant. This replaced ammonium and potassium oxalates
double salts. This anticoagulant is used as tri-potassium or dipotassium or disodium salt. Liquid
dipotassium is preferred over tri-potassium salt because of its solubility and because it is
associated with fewer artefactual changes.
Note:Dipotassium salt is very soluble i.e. 1650g/l-- This is the reason as to why it’s preferred to:
Disodium salts –108g/l
Dilithium salts –160g/l
EDTA is the anticoagulant of choice for:-Morphological testing of blood cells, Blood cell
counting, Haemoglobin estimation test, etc
Mode of action of EDTA anticoagulant
This anticoagulant prevents clotting of blood by chelating calcium ions or binding calcium ions.
In the laboratory, EDTA can be provided at a concentration of 7.5% or 10%.The EDTA
anticoagulant recommended by the international council for standardization in haematology is
dipotassium at a concentration of 1.50 ± 0.25mg/ml of blood. At this concentration, tris-
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potassium salt produces some shrinkage of the red blood cells which results into a 2-3% decrease
in the PCV.
DETERMINATION OF THE CONCENTRATION OF EDTA THAT CAN PRESERVE A
SPECIFIC VOLUME OF FRESHLY COLLECTED BLOOD
Assuming you have been provided with 7.5% EDTA salt.
Approach
7.5 g of EDTA are dissolved in 100mls of distilled water.
Converting grams into milligrams
1g = 1000mg
7.5 x1000mg of EDTA is contained in 100mls of distilled water.
This is the same as
100mls of distilled water contains 7500mg of EDTA
1ml of distilled water contains 7500x1 = 75mg of EDTA
100
If 1ml of distilled water contains 75mg of EDTA,
Xmls of distilled water will contain 1.5+_0.25mg of EDTA
X = (1.5x1) mls of water
75 = 0.02mls
0.02mls of distilled water contains 1.5mg of EDTA therefore 0.02mls of EDTA solution
preserves 1ml of blood.
Procedures
Place 0.02 mls of EDTA into a dry clean bijou bottle (0.02x1000 = 20microliters of EDTA
preserves 1ml of blood).Warm in the incubator to evaporate the water
Label the tube or the bottle
The following information should be included on the label
Volume of blood to be preserved, date of preparation
Anticoagulant, concentration of the anticoagulant, the one who has prepared the bottles.
Question: Assuming you have been provided with EDTA stock solution of 10.0g/dl, describe
how you would prepare bottles to preserve 5mls of freshly collected blood?
Advantages of using EDTA anticoagulant.
This anticoagulant preserves the morphology of blood cells
It prevents the formation artefactual changes when the blood specimen is stored for up to 3
hours. This anticoagulant can be used to make blood films
Note :There are negligible changes when dipotassium salt is used at the correct concentrations
but when excess of this anticoagulant is used or when the sample is left to stand for long before
processing, then this can result into artefactual changes. Excess of EDTA irrespective of which
salts affects RBCs, platelets and WBCs causing:-Degeneration, Shrinkage changes, etc
WBCs
Changes occur mainly after 3hours of blood collection. They include:-Swelling and loss of
structure in the nuclear lobes for example there is stretching of the inter-lobular bridges.
Loss of Cytoplasmic granulation
Vacoulation in the nucleus and in the cytoplasm.
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There may be cell disintegration leaving a bare nucleus.
The nucleus of the white blood cells stains homogenous.
Platelets
Excess anticoagulants cause platelets to swell and finally disintegrate, this can result into
elevated platelet counts because the fragments can be counted or mistaken to be platelets.
Also on rare occasions EDTA can cause the platelets to clump.
Red blood cells
After about 6 hours, red blood cells will begin to swell thereby causing an increased MCV,
decreased ESR and increase in osmotic fragility values. There is Anisocytosis, spherocytosis,
Macrocytosis and Haemolysis of the red cells
Note: The older the specimen, the more will be the morphological changes.
The following should be ensured to prevent occurrence artefactual changes
Accurate amounts of the anticoagulants should be used.
Not allowing the samples to stand for long before processing
Thorough mixing of the sample with the anticoagulant.
TRISODIUM CITRATE ANTICOAGULANT
This is the anticoagulant of choice when performing coagulation and ESR tests. It helps to retain
the platelet functional abilities and viability of coagulation factors. It is provided in two
concentrations namely:-
3.8 %( 129mmole /L)
3.2 %( 109mmole/L)
This can be given as arrange as below.
100 –120mmol/l tri-sodium citrate (32g/l)
Na3C6H5O7.2H2O
MODE OF ACTION OF TRI-SODIUM CITRATE ANTICOAGULANTS
This removes the calcium by loosely binding with it to form a citrate complex.
When using this anticoagulant for coagulation tests, 9parts of whole blood and 1part of the
anticoagulant are mixed. Also it is the anticoagulant of choice for the erythrocyte sedimentation
rate test where 4parts blood and 1part of the anticoagulant are mixed.
Note: For transfusion purposes, citrate is mixed with phosphates, adenine and dextrose for long
term storage.
HEPARINE ANTICOAGULANT
This is available as lithium, sodium, potassium and as ammonium salts. It is said to naturally
present in the body and it is used at a concentration of 15-30IU /ml of whole blood .but most
tubes are prepared with 0.2mg heparine /ml of blood. It is prepared by dissolving 0.4g of the
powder into 100mls of distilled water.
Note: Heparine anticoagulant is used when we intend to perform the following tests:-Osmotic
fragility test, electrolyte estimation and Red cell enzyme estimation tests.
Mode of action of anticoagulants
Antihuman thrombin iii is an inhibitor of activated coagulation factors. I.e. 11, X, IXa, Xa, X1a,
XIIa, XIIIa and others. Heparine accelerates the action of antithrombin iii which neutralizes
thrombin, thus preventing the formation of fibrin from fibrinogen. Heparine is also present
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naturally in blood. Most tubes are prepared with 0.2mg heparine of blood collected. The
advantages of using heparine anticoagulant is it does not alter the size of the red blood cells
Note: It is of limited use in haematology because blood smears prepared from heparinized blood
when stained will have a blue back ground particularly in presence of abnormal proteins and it
has a temporary action
Disadvantages associated with heparine anticoagulant
Heparine can cause leucocyte clumping hence it cannot be used when performing the WBC total
counts.
OXALATES: These acts by precipitating calcium ions. The calcium can be precipitated in the
none ionized forms.
Effects of storage of blood on cell morphology
Morphological changes occur on blood cells when the blood sample is left to stand for long after
collection.
Note: If the films are made within 3hours of blood collection, normal changes occur. Beyond 6
hours after blood specimen collection, the following changes occur:-Shrinking of the neutrophils
Neucleus of neutrophils stains more homogenously than in freshly collected blood samples.
The nuclear lobes may be separated
The Cytoplasmic margin may appear ragged or less well defined.
Vacuoles may appear in the cytoplasm.
Changes in Monocytes
Small vacuoles appear in the cytoplasm. There is irregular lobulations of the nucleus. This can
lead to disintegration. The nucleus may be more homogenous than usual.The nucleus undergoes
budding giving rise to nuclei with 3 lobes. These artefactual changes may not be distinguished
from apoptosis.
Note: Normal red blood cells are little affected by standing the blood specimen for up to 6 hours
or for longer periods. Some of the changes include:-
Crenation of the red blood cells
Sphering of the red blood cells can result.
A table showing the different blood collection vacutainer, their colour codes and tests
performed on such blood specimens
Anticoagulant used Colour of the Examples of the tests performed on the sample.
vacutainer
EDTA anticoagulant Purple Hb estimation, preparation of blood films, cell counts, etc.
Sodium citrate Light blue Coagulation tests and ESR test.
anticoagulant
Heparine anticoagulant green Osmotic fragility test.
Enzyme estimation tests
Fluoride oxalate grey Routinely used in the chemistry tests when estimating the
anticoagulant glucose levels
Plain vacutainer red Cross match tests and compatibility tests .etc.
References
Monica Cheesbrough. (2000).District laboratory practice in tropical countries part two
Baker. F, J and R, E, Silverton. (1998) introduction to medical laboratory technology (Ed) 8.
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Dacie and Lewis (2001 –2002). Practical haematology. Ninth edition.
Arthur Simmons (1997).combined theoretical and practical/ technical approach. 2 nd edition.
