Purpose Of Anticoagulants

Blood is a combination of formed elements (RBCs, WBCs, Platelets) in a liquid portion

called plasma.
These are used to prepare the whole blood or plasma during the collection of the blood
samples.
There is a difference in the plasma and the serum for the estimation of various
substances in the blood.

Serum and plasma difference

Indications
A whole blood sample is used for blood gases and ammonia.
It may be used for glucose, urea nitrogen, and lactate estimation.
Serum and plasma are used for the majority of the chemical tests.
The disadvantage of plasma is if you store the sample then there are chances for the
formation of fibrin clots.
These microclots may block the probe of the analyzer.
Plasma is not a good sample for electrophoresis.
Type of patients from whom you are going to get blood samples are:
Pediatric patients: If this is the first time sample from the child, then take time to gain
his confidence.
Blood for neonatal screening is collected to rule out hypothyroidism, phenylketonuria,
galactosemia, and hemoglobinopathies.
For phenylketonuria, take the blood at least after 24 hours and the infant has taken the
feed.
Adult patients: Be friendly and explain the procedure.
Patients in ICU and who are unconscious: No doubt the patients are unconscious but you
should take care of taking the blood samples.

Types of the blood sample

Type of collection procedures:
Capillary blood (skin puncture).
This is good for a small quantity of blood.
Warm the finger taking the blood sample.
In the newborn, under 3 months the heel is the best site to get a small quantity of blood.
The depth should not be >2.4 mm on the heel.
Avoid the central portion and back of the heel.
Venous blood (venipuncture):
For larger quantities, venous blood will be taken.
The blood sample is taken from the forearm, wrist, or ankle veins.
A forearm site is preferred. Blood is taken directly from the vein, called phlebotomy.
The median cubital vein is usually preferred.
Mostly venous blood is drawn in the fasting state.
Blood collected after the meal is called a postprandial sample.
There are biological variables in the blood collection like:
Patient lying in the bed or standing up.
After the exercise.
Diurnal variations.
Recent food intake.
Recent intake of Tea/coffee (caffeine), smoking (nicotine), alcohol ingestion, and
administration of the drugs.
A blood sample can be taken in vacutainers, syringes, and butterfly needles.
The blood samples can also be taken for blood culture.

Arteria blood is needed for the blood gases.

Arterial blood is usually taken from the femoral artery.
Blood for gases should be processed immediately without any delay.
In Routine Used Anticoagulants Are:
EDTA (Ethylenediaminetetraacetic Acid)
EDTA is used as a disodium or dipotassium salt.
Mostly potassium EDTA is used as an anticoagulant,
recommended for hematology studies.
This is a chelating agent that binds the calcium which is
needed for coagulation.
It is effective at a final concentration of 1 to 2 mg / mL
of blood.
This can be used as a powder or make the solution and
then add to vials. Let it dry.
It is used as disodium, or dipotassium or tripotassium
salt.  
Solution:
EDTA solution of 0.1% can be prepared and used. Let it evaporate at room temperature.
Or 1.5 mg/mL.
More than 2 mg/mL causes shrinkage of the cells.
It is used for:
This is the choice anticoagulant in the hematology section.
It is used for cell count, hematocrit, hemoglobin estimation, and the cell differential
count.
Advantages:
EDTA preserves the morphology of the blood cell structure.
This is the anticoagulant of choice for hematocrit, Hb, and differential count.
This is the best anticoagulant for peripheral blood smear and studies.
 It has little effects on the various tests.
They produce less shrinkage of RBCs.
There is less increase in the cell volume after keeping the blood.
Drawbacks:
It inhibits the alkaline phosphatase, creatine kinase, and leucine aminopeptidase
activities.
EDTA is not suitable for Calcium and iron
estimation.

EDTA as an anticoagulant
Heparin
This is an anticoagulant and causes the
least interference with the test.
This is theoretically the best anticoagulant
because it is a normal component of the
blood and does not introduce any foreign
contaminants to the blood specimen.
This acidic, mucopolysaccharide with a molecular weight of 15,000 to 18,000, is a blood
coagulation inhibitor by potentiating the activity of the antithrombin.
This is more costly than the others.
It is present in powder form but is hygroscopic and dissolves rapidly.
It is mucoitin poly sulfuric acid available as sodium, potassium, lithium, and ammonium
salts.
Mechanism of action:
It is not absorbed by the GI tract, so given by injection in case of therapy.
Heparin accelerates the action of antithrombin III   which neutralizes thrombin
thus prevents the formation of fibrin from fibrinogen.
It forms the complex of thrombin + antithrombin cofactor + heparin and prevents fibrin
clot formation.
It prevents the coagulation for 24 hours by neutralizing the thrombin, thus preventing
the formation of fibrin clot from the fibrinogen.

Heparin is an anticoagulant and its mechanism

Solution:
Heparin is added 0.2 mg / mL of blood in each
test tube.
Or 20 units of heparin for 1 mL of blood (in
another reference 15 U/mL).
or a drop of heparin is drawn into the syringe.
Or simply coating the inside of the tubes or
syringe is enough for the anticoagulant effect.
After the collection of blood inverts the tubes for 5 to 7 times for proper mixing of the
blood.
Advantage:

 This is the best anticoagulant to use dry when minimal hemolysis is desired e.g. for
sodium and potassium estimation.
This is the best anticoagulant used for the estimation of pH, blood gases, electrolytes,
and ionized calcium.
Drawback

It is costly.
It inhibits the acid phosphatase activity.
It gives a blue background for Wright’s stain
smears so not good for peripheral blood smear
interpretation.
It also affects the binding of triiodothyronine and
thyroxine to their carrier protein and produces a
higher free concentration of these hormones.
It interferes with the binding of calcium
to EDTA.
It is not used for coagulation and hematology studies.
Ammonium heparin has an effect on the RBCs volume.

Sodium Citrate
Citrate is used as trisodium citrate salt.
It is a white hygroscopic crystalline powder.
Purpose:
Sodium citrate is widely used for coagulation studies.
For PT and PTT.
The sample can be used for ESR by the
Westergren method.
Mechanism of action:
it is used in solution form.
This will chelate calcium. Inactivates
Ca++ ions.
This will prevent the rapid deterioration of labile coagulation factors like factor V and
factor VII.

Solution preparation and uses:

Trisodium citrate= 3.2 to 3.8 g/dL (3.2% solution).
Mix well Trisodium citrate 3.8 grams in distle water.
This can be used as 0.109 mg/mL.
In blood, its ratio is 1:9 where 9 parts are blood and 1 part is sodium citrate.
PT and PTT= Blood: Sodium citrate = 9: 1 part (blood 9 parts: sodium citrate 1 part)
ESR = Blood: Sodium citrate = 4:1  (1.6 mL of  blood: o.4 mL Sodium citrate).
Drawbacks
This is used in liquid form (liquid anticoagulant).
This is not a good anticoagulant for a complete blood examination.
This is not good for the estimation of calcium.
It inhibits aminotransferase and alkaline phosphatase.
This will stimulate acid phosphatase when phenyl phosphate is used as the substrate.
It has little value in clinical chemistry.
Potassium Oxalate
This may be sodium, potassium,
ammonium, or lithium oxalic acid salt used
as an anticoagulant.
This form insoluble complex with calcium
ions (precipitate with calcium as a salt).

This is the most popular oxalate salt used

as an anticoagulant in powder form.
Solution:
Potassium oxalate at a concentration of 1
to 2 mg/mL of blood is used.
Mix 30 grams/dL in distal watr.
Now add a few drops in the test tube side and dry it in the oven below 100 °C.
The combination of ammonium/potassium oxalate does not lead to shrinkage of the
RBCs.
While other oxalates cause shrinkage.
Drawbacks
If the concentration is >3 mg/mL, then there are chances for hemolysis.
There is a reduction of 10% hematocrit.
Oxalates inhibit several enzymes like acid phosphatase, alkaline phosphatase,
amylase, LDH.
It may cause precipitation of calcium as oxalate salt.
Sodium Fluoride

This is a weak anticoagulant but used

an antiglycolytic agent to preserve
the glucose.
This inhibits the system involved in
glycolysis and preserve the glucose.
This can be used as a dry additive.
Mechanism of action: It acts in two
ways:
As an anticoagulant by binding the
calcium.
As an enzyme inhibitor which
prevents the glycolytic enzyme to
destroy the glucose.

Solution:
This is effective at a concentration of 2 mg/mL of blood along with another
anticoagulant like potassium oxalate.
When used alone then more concentration than 2 mg/mL is needed.
This can be used in combination with oxalate as a fluoride-oxalate mixture.
Most specimens are preserved at 25 °C for 24 hours and at 4 °C for 48 hours.
Sodium fluoride is poorly soluble so mix blood thoroughly before effective anti-glycolysis
occurs.
This is mostly used for glucose estimation.
Drawback
This is also an inhibitor of many enzymes.
Also, effect urease for the estimation of urea.
Sodium Iodoacetate

This is also an antiglycolytic agent at a concentration of 2 g/L.

This may be substituted for sodium fluoride.
This has no effect on urease.
Drawback:
It inhibits creatine kinase.
Adverse effects of the additives:
The additive may contain the substance to be tested like Na +oxalate for the estimation of
Na+.
The additive may remove the component to be tested like in oxalate, removes the
calcium.
The additive may affect enzymes like Na+flouride. This may destroy many enzymes.
A small amount of the anticoagulant gives rise to microclots and this will interfere with
cell count.
The additive may distort the cells like oxalate will change the cell morphology like RBCs
and these will become crenated. While WBCs show vacuoles. Lymphocytes and
monocytes will have distorted shapes.
If the excess quantity is used that will dilute the substance to be tested.
Various Blood Samples:
Whole Blood
This blood sample obtained in the test tube containing an anticoagulant.
This sample will contain cells (white blood cells, platelets, RBCs,
proteins) and plasma.
Plasma
This is a pale yellow liquid which contains RBCs, white cells, and
platelets.
Plasma forms with the help of anticoagulants which will prevent
the clotting.
There is the presence of fibrinogen in the plasma.

plasma constituents
Serum
This is a clear fluid that is separated from the clotted blood. There
are no RBCs, white cells, or platelets. There is no need for anticoagulants.
Clotted blood is kept at 37 C for at least 20 minutes and then centrifuged.
The upper portion is called serum.
There is no fibrinogen.

Serum and plasma difference

Plasma contents Serum contents

Contains all proteins (albumin, globulins, and Fluid remaining after

fibrinogen) coagulation

 Contains fibrinogen No fibrinogen

90% of water (92 to 95%) 90% of water

RBCs, WBCs, and platelets are suspended in plasma No RBCs, No WBCs, No

platelets

Electrolytes same level Electrolytes same level

No prothrombin

Antibodies are present Antibodies are present

 Gases (CO2, O2, and N2) No clotting Factor VIII, V, XIII

 Glucose, amino acids, cholesterol, and fats Contain factor XII, XI, X, IX, VII

 Hormones Contain rest of all products like

plasma

 Excretory products like urea, uric acid, creatinine, and Excretory products present
bile products

The same value of bilirubin, cholesterol, and creatinine The same value of bilirubin,
cholesterol, and creatinine

The difference between plasma and the serum

 Characteristics Plasma Serum

Fibrinogen 0.2 to 0.4 G/dL Nil

Formation site Present in the body fluid Prepared outside the body

Outside the body Always contains anticoagulant Never anticoagulant added

contains

Chemical substances Plasma values more Plasma values less than No difference in the value in serum
than serum serum and plasma

Calcium 0.9%

Chloride 0.2%

Total protein 4%

LDH 2.7%

Albumin 1.3%

SGOT 0.9%

Alkaline phosphatase 1.6%

glucose 5.1%

Bicarbonate 1.8%

Sodium 0.1%

Phosphate 7%
Potassium 8.4%

Urea 0.6%

Uric acid 0.2%

Bilirubin

Creatinine

Cholesterol

The following table elaborates on the difference between plasma and serum as regards
the values of constituents of blood.

Buffy Coat
This is the middle layer between the plasma and RBCs.
This will contains white cells and platelets. .
Stopper Additives Outcome of additive Purpose of use Test
tube tubes

Red None For serum 1. Chemistry

2. Serology

3. Blood banking

Lavender EDTA 1. Anticoagulant, for 1. CBC

plasma
2. CEA
2. Remove calcium and
prevent clotting 3. Best for
hematology

Orange Thrombin 1. Accelerate clot 1. All serum test

usually in 5 minutes,
for serum formation 2. Chemistry

2. Should be inverted 8
times

Light Sod. citrate 1. anticoagulant binds 1. Coagulation

Blue calcium. studies

2. Get blood or plasma 2. PT, APTT, factor

assay

Gray Na fluoride/K oxalate 1. Inhibit glycolysis 1. Glucose

2. Anticoagulant, 2. GTT
remove Ca++ to
prevent clotting 3. Lactate

3. Get whole blood or

plasma
Green 1. Na+ Heparin 1. Inhibit thrombin 1. Ammonia,
activation to prevent
2. Lithium Heparin clotting. 2. Carboxy Hb

3. Ammonium 2. Get whole blood or 3. Lead

Heparin plasma
4. Plasma chemistry

Yellow Na+polyanetholesulfate 1. Prevent blood from Blood culture

clotting

2. Stabilize bacterial
growth

3. Invert the tube 8

times to prevent
clotting

Glod 1. Gel-separator 1. Clot activator shorten 1. Most chemistry

the time for clot tests
2. Clot activator formation
2. Not good for blood
2. The gel forms a banking
separator between
cells and serum

3. Invert tube 5 times

and centrifuge after
the clot formation

Light 1. Gel separator 1. Heparin prevents Potassium determination

green clotting
2. Lithium heparin
2. The gel prevents
cells contamination

Black 1. Na + citrate 1. Binds  Ca++ Westgreen ESR

 
2. 4:1 ratio of blood to
anticoagulant

olor coding for the blood samples: