UNIT 2

ANTICOAGULANTS
 Anticoagulants
Introduction:
• Concept of anticoagulants was stared form blood banking
purpose.
• First anticoagulant preservative was introduced by Rous
and Turner in 1916 consisting of citrate-glucose solution.
• Rous Turner’s solution was used for human blood storage
during first world war.
• Later on with the development of technology, it was started
to obtain non-clotted blood specimen mainly for
hematological studies.
Definition:
• Anticoagulants are the Chemical substances or agents that
prevent the blood from clotting when mixed with blood in
appropriate concentration either by chelating calcium ions
or inhibiting many steps necessary for coagulation of blood.
• Sometimes they also acts as preservatives.

Purpose of studying anticoagulants:

In vivo (inside body)
For the therapeutic purpose
In vitro (outside the body)
• For study of the various constituents of blood components.
• Study of blood coagulation(clotting)
• Preservation of blood in blood bank.
Characteristics of Anticoagulants
An anticoagulant selected for use in hematological
examination must have the following qualities:
1. It must not alter the morphology of the cell.
2. It must not cause hemolysis.
3. It must minimise platelest aggregation.
4. It must minimize disruption of staining and morphology
of leucocytes.
5. It must be readily soluble in water.
6. It must be soluble un blood.
7. It must be keep the blood in fluid condition.
 Types of Anticoagulants
A. Calcium Chelators B. Non- calcium chelators
1. Oxalates 1. Heparin
i. Potassium oxalate 2. warfarin
ii. Ammonium oxalate
iii. Double oxalate
2. EDTA
3. Tri-sodium Citrate
4. Sodium fluoride
WORKING PRINCIPLE OF ANTICOAGULANT
• Most of the anticoagulant commonly used acts by
removing the calcium ions present in the blood which is
required for coagulation process.
• The anticoagulant binds with the calcium and thus
prevents blood from clotting. Eg: calcium chelators
• Also, some anticoagulants such as sodium fluoride which
inhibits the activity of Phosphorylase enzymes and provides
precise results in Blood Sugar Estimation.
• Some anticoagulants prevent blood clotting by inhibiting
prothrombin formation, thromboplastin formation and
various other clotting factors formation or their activator.
Eg: Heparin, warfarin,etc.
A. Calcium chelators
• When blood is mixed with anticoagulant they bind with
calcium (Ca++)present in blood and form insoluble salt or
soluble but unionized salt of calcium.
• As calcium is the key factor for blood coagulation, when it
is precipitated by chelators, coaguation is prevented.
• This prevents clotting once calcium are essential for many
of the steps in the coagulation mechanism.
• Different types of calcium chelators are explained below:
1. Oxalates
• Oxalates combines with the calcium in the blood to form
an insoluble calcium oxalate compound, which precipitate.
• Oxalates are found as potassium and ammonium oxalates
or mixture of these oxalates.
i. Potassium oxalate
• This is used at a concentration of 2mg per ml of blood.
This anticoagulant is mostly used for chemical analysis.
• Potassium oxalates shrinks RBC’s (about 8%). Therefore it
is not used for the tests like PCV, ESR and for peripheral
blood smear.
ii. Ammonium oxalate
• It is used as a concentration of 2mg per ml of blood.
• This anticoagulant causes swelling of RBC’s. Therefore it is
also not used for the tests like PCV, ESR and for peripheral
blood smear.
• The calcium in the blood combines with oxalate to form
insoluble calcium oxalate.
iii. Double oxalate
• Oxalates that are found as potassium and ammonium
oxalates or mixture of these oxalates are known as Double
oxalates.
• Potassium oxalate or ammonium oxalate are used together in
the ratio of 2:3 (0.8mg and 1.2mg ) this is done to counter
the swelling effect of ammonium oxalate and shrinkage effect
of potassium oxalate on the RBC.
• Double oxalated blood may be used for the hemoglobin
estimation, PCV, WBC, RBC, Prothrombin time, ESR test by
wintrobe’s method.

Oxalates – Mechanism of Action

• It acts as a chelating agent and binds with the calcium ions
present in the blood and forms insoluble precipitates of
Disadvantages of Oxalates
1. These anticoagulants are not used in the chemical
analysis.
2. It donot preserve the morphology of the cells.
3. This anticoagulant is toxic not to use for the injection.
4. Potassium oxalate causes shrinkage and ammonium
oxalate causes swelling of RBC’s therefore they are not
recommended for blood smear, PCV, ESR test.

Uses of double Oxalates

It can be used for the Blood chemistry, Packed cell volume
(PCV), Erythrocyte Sedimentation Rate (ESR), Total
Leukocyte Count (TLC), Specific gravity etc.
Preparation of Double Oxalate
• The double oxalate consist of
• Ammonium Oxalate – 1.2 gm (3 parts)
• Potassium Oxalate – 0.8 gm (2 parts)
• Water – 100 ml
• It as prepared as the 20 mg/ml concentrated oxalate solution
that means 2mg/0.1 ml of oxalate. 0.5 ml of this solution is
poured into the container and dries, which is sufficient for 5 ml
of blood.
• Remember that the Potassium oxalate and Ammonium Oxalate
should be used in a ratio 2:3 and at a concentration of 2mg/ml
of blood.
Advantages of Double oxalates
• Double oxalate is preferred as it prevents the swelling effect
of Ammonium oxalate & shrinking effect of Potassium
oxalate on the RBCs.
EDTA (Ethylene Diamine Tetra acetic acid)
• This is used in 3 forms:
i. Disodium salt of EDTA (Na2 EDTA)
ii. Dipotassium salt of EDTA(K2 EDTA)
iii. Dilithium salt of EDTA (Li2 EDTA)
• The commercial name of EDTA are Versene (Na2 EDTA) and
Sequestrene (K2 EDTA).
• Among them Dipotassium salt of EDTA is preferred because
K2 EDTA is more soluble than others.
• It is widely used chemical anticoagulant in the laboratory.
• EDTA is colorless, water soluble and acts as a most powerful
calcium chelating agent.
• It has the ability to bind to metal ions like calcium and
change it into soluble but unionised form.
• EDTA are used in the concentration of 1-2 mg per ml of
blood.
• It gives the best preservation of cell morphology and it is
therefore preferred anticoagulant for all cell count and
smear.
• Smears can be made upto 3 hours after collecting blood
and good morphology is still preserved.
• Platelet clumping is inhibited, so it is good anticoagulant for
platelet count also.
• Blood collected in EDTA can be used for TLC, PS
preparation, Hemoglobin Estimation and Differential
Leukocyte Count (DLC).
Mechanism of action of EDTA
• It acts as a chelating agent. It has the ability to bind to
metal ions like calcium and change it into soluble but
unionised form. (calcium is required for blood to clot, so
when it is removed blood will not clot).
Advantages of EDTA
• It gives better preservation to the cellular morphology of
blood cells when observed even after 3 hours of blood
collection.
• It can be used for platelets counting as it inhibits the
clumping of platelets.
• EDTA blood sample can be stored overnight at 4 degree C
without detoriation.
• Preferred anticoagulant for all cell count and smear for DLC.
• Most important advantage is that EDTA is readily soluble.
Disadvantages of EDTA
• Excess of EDTA in the blood may lead to shrinkage of RBCs &
WBCs. It may cause degenerative changes in the blood cells.
• Also, the excess amount of EDTA may cause the decrease in
Packed Cell Volume (PCV) & Increase in MCHC (Mean Cell
Hemoglobin Concentration).
• It activates naturally occurring anti-platelet auto-antibodies
which cause the platelet adherence to Neutrophils.
• Not suitable for blood coagulation studies as it chelate
calcium.
• Not suitable for blood transfusion and estimation of
calcium in serum.
• It fails to demonstrate basophilic stippling of red cells in
lead poisoning.
• Not suitable for electrolyte estimation.
Uses of EDTA
Following tests are commonly done by using EDTA as an
anticoagulant –
• Complete Blood Count (CBC)
• Hemoglobin estimation
• Hematocrit or Packed Cell Volume estimation
• ESR by wintrobes method
• HbA1C test
• Platelet count
• Red cell Indices
• Differential Leukocyte Count
Preparation of EDTA vials/tubes
10% EDTA solution is prepared in the laboratory
• 10gm of EDTA is dissolved in 100ml of distilled water.
• Pour 20 µl of above prepared solution in the individual vial.
• Evaporate the vials in the incubator or room temperature
leaving the white powder at the bottom of each vial ( for
quick evaporation, 60˚C in hot air oven is used).
Calculation:
10% of EDTA in distilled water:-
10gm EDTA ------100ml distilled water
10000mg EDTA ------100000 µl distilled water
1mg EDTA-----------10 µl distilled water
2mg EDTA in ---------20 µl distilled water
 Tri-Sodium Citrate
• Trisodium citrate is a liquid anticoagulant. It was first used
as an anticoagulant in blood transfusion in the earlier
20th century.
• Its usefulness continues till today in blood collection
tubes for the ESR and Coagulation studies as well as for
the preservation of blood in blood banks.
• 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.
• 3.8% tri-sodium citrate is commonly used in laboratory.
• For coagulation testing, the ratio of 9 volumes of blood to
one volume of anticoagulant (9 volumes blood: 1 volume
anticoagulant) is 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).
• Used as a liquid, so dilution of cellular elements occurs
making it undesirable for counts, Hb estimationétc.
Tri-Sodium Citrate – Mechanism of Action
• The action of Citrate ions is to form calcium citrate
complexes which disrupt the blood clotting mechanism by
chelating or binding with the calcium ion in unionised but
soluble complex form.
Preparation of Tri-Sodium Citrate
• 3.8% solution of Tri-sodium citrate is commonly used
which can be easily prepared in laboratories as follows:
• Tri-sodium Citrate – 3.8gm
• Distilled Water – 100ml
• Dissolve the Tri-Sodium Citrate in Distilled water and mix
well. 0.4ml of this anticoagulant is sufficient for the 2ml of
blood.
Uses of Tri-Sodium Citrate
The Citrated blood is used for
• ESR estimation by Westergren Method – for 1 volume of
citrate, 4 volume of blood is added.
• Coagulation studies (PT, APTT) –1 volume of citrate, 9
volume of blood is added. (1.8ml blood in 200µl sodium
citrate)
Disadvantages:
• Citrated blood cannot be used for Packed Cell Volume
(PCV), Hemoglobin (Hb) Estimation, Total Leukocyte Count
TLC, and Differential Leukocyte Count (DLC) because
citrate is used as a solution and it alters the concentration
of blood.
• Donot preserve the cell morphology well.
SODIUM FLUORIDE
• It is the anticoagulant of choice for the estimation of blood
sugar.
Sodium Fluoride – Mechanism of Action
• It binds with the calcium ions present in the blood and
forms Calcium fluoride.
• Also, it is commonly used whenever the blood sugar
estimation is required to be done because Sodium Fluoride
is the competitive inhibitor of Phosphorylase enzymes and
prevents glycolysis by blocking its activity.
Preparation of Sodium Fluoride
• It is commonly used at the concentration of 6mg/ml of
blood and can easily be prepared in the laboratory as
follows –
 Sodium Fluoride – 3gm
Distilled water – 100 ml
• Dissolve the content and mix well. It gives the solution at a
concentration of 30mg/ml.
• 1 ml of this solution is poured into a container and allowed
to dry, which is sufficient for 5 ml of blood as 6mg/ml is
used.
Uses of Sodium Fluoride
• It is commonly used for the estimation of Blood Sugar and
other biochemical tests.
B. Non- Calcium Chelators
1. BIOLOGICAL / NATURAL ANTICOAGULANT – HEPARIN
• It is mucoitin polysulphuric acid.
• Heparin is a highly acidic substances which is normally
present in the body in small amount.
• Heparin is a natural anticoagulant which cannot be
prepared in the laboratory.
• It is available as Na, K, Lithium, NH4 salts as a dry powder,
hygroscopic in nature and dissolves rapidly.
• It is a good anticoagulant and well preserves the
morphology of the Red Blood Cells (RBCs).
• It is used in the concentration of 10-120 IU/ml equivalent to
0.1-0.2 mg/ml of Blood.
The mechanism of action
• The mechanism of action of heparin is (Anti- thrombin
III)ATIII-dependent.
• It acts as an inhibitor of thromboplastin formation and
secondarily as an anti-thrombin i.e, inhbiting the
interaction of several clotting factors in the presence of a
plasma co-factor anti thrombin III (a major coagulation
inhibitor) i.e, inhibit the action of the thrombin or
fibrinogen.
[Note: heparin inhibits but doesnot destroy and therefore
can be neutralized by another compound protamine.]
 Uses:
• It can be used for the osmotic fragility tests.
• For electrolyte estimation in biochemistry.
• For anticoagulation of some blood in blood bank,
particularly for open heart surgery.
• Heparin is most widely used anticoagulant for urgent
clinical chemical analysis.
• Immunophenotyping.
Advantages:
1. Best anticoagulant for open heart surgery.
2. Doesnot alter the cell morphology especially RBC.
3. Instanteous anticoagulation.
4. Minimum hemolysis.
Anticoagulants used for transfusion purpose in blood
bank
Criteria for selection of anticoagulants
• Should efficiently prevent blood clotting
• Should not be toxic and produce deleterious effects on
recipient.
• Should not alter any of the functions of components of
collected blood: maintain cell viability.
• Should help to increase stability of collected blood when
stored in refrigerator.
Anticoagulants used are:
Sodium citrate is most commonly used anticoagulant but it
will not preserve blood for long time. Thus mixture of
solution is used;
1. Acid-Citrate Dextrose (ACD)-----28days storage
2. Citrate-Phosphate Dextrose(CPD)-----28days
3. Citrate-Phosphate Dextrose Adenine (CPDA)-----35days
4. Citrate-Phosphate Dextrose Adenine-1 (CPDA-1)-----35days
1. Acid Citrate Dextrose (ACD)
• Acid Citrate Dextrose (ACD), also known as Anticoagulant
Citrate Dextrose Solution is used as an anti-coagulant for
whole blood and erythrocyte survival, routinely used for
blood storage.
• It is a sterile, non-pyrogenic solution of citric acid, sodium
citrate, and dextrose, in water.
2. Citrate-Phosphate Dextrose(CPD)
• Gibson (1957) showed that by adding inorganic phosphate
buffers to ACD, the post transfusion survival rate of Red
cells could be increased i.e; red cells viability.

CPD solution:
Trisodium citrate dihydrate-26.30gm
Citric acid (monohydrate)-3.27 gm
Sodium dihydrogen phosphate(monohydrate)-2.28gm
Distilled water-1 litre
• 14ml of the solution is used for 100ml of blood. The blood can
be stored for 28 days.
• CPD requires less citric acid than ACD. It is better than ACD due
to slightly higher pH and because of phosphate compound.
• The survival of red cells, 24 hours after transfusion is about
80% in CPD solution compared with 70% in ACD solution.
Stored blood for 24 days.
• CPD contains enough dextrose to support continuing ATP
generation by glycolytic pathway.
• Red cell 2,3-DPG (Disphosphoglycerate)is better maintained in
CPD solution than ACD; with ACD 2,3 DPG is lost from blood at
the end of the week wheras 2,3 DPG is better maintained in
CPD and is not depleted for about 2 weeks.
• Sodium- for electrolyte balance
• Phosphate- for phosphorylation i.e: ADO or AMP to ATP..
3. CPDA-1 (Citrate Phosphate Dextrose Adenine -1)
• Latest recommendation of blood storage for blood.
• Adenine allows red cells to maintain the adenine nucleotide
pool providing substance for ATP synthesis.
• Human RBC does not contain enzyme to synthesize adenine
and other purines.
• Storage time- 35days
• With adenine adequate levels of 2,3-DPG can be
maintained for 12-14 days.
4. CPDA-2
• In CPDA 2 amount of adenine is increased to 0.55gm and
dextrose to 44.6gm.
• It is better than CPDA-1 due to more dextrose and adenine.