BLOOD COLLECTION

METHODS &
ANTICOAGULANTS
Department of Biotechnology, V.N.S.G.U., Surat

Milan R. Patel
Assistant Professor, DBT VNSGU, Surat
UGC-CSIR-NET
• Blood sampling or blood collection method is an essential procedure
in modern medicine.
• The blood can be taken from a vein where it is called venous blood
collection or arteries where it is called arterial blood collection.
• Minute quantities of blood can be taken from various sites by pricking
the skin.
TECHNIQUE OF BLOOD COLLECTION :
• Blood is collected from the vein for various hematological investigations. In
order to obtain accurate and precise results in the laboratory which will help
the clinician to make a correct diagnosis of the patient’s disease, it is of
paramount importance to collect the blood sample in a correct manner.
• Each sample is sent to the laboratory accompanied by a laboratory
requisition form filled in by the clinician.
• Brief clinical details and any other relevant information must be mentioned
on the form. Prior to blood sample collection it is essential to check the
patient’s identity and make sure that it corresponds to the name and other
details mentioned on the requisition form.
• Blood can be withdrawn from the vein, usually the antecubital vein on the
forearm (Venous blood) or from the finger or heel (Capillary blood).
• Venous blood is preferred. It can be collected using a syringe and needle or
a vacuum tube. Both these methods will be described separately.
COLLECTION OF VENOUS BLOOD :
• Blood is usually withdrawn from the antecubital vein or any other vein
which is well identified on the forearm. The vein selected should be large,
readily accessible, and sufficiently close to the surface to be seen and
palpated.
• Preparation of venipuncture site Clean the skin of the area around the
identified vein with 70% isopropyl alcohol in a circular fashion beginning at
the site and moving outward.
• Allow to dry spontaneously. Do not touch the venipuncture site after it has
been cleaned. Apply a tourniquet 3-4 inches above the venipuncture site.
Ask the patient to make a fist a few times. Veins suitable for puncture will
then become more apparent.
• Veins can become distended and easier to enter by allowing the arm to hang
down for 2 or 3 minutes or by gently slapping the site of puncture.
COLLECTION OF VENOUS BLOOD USING A
SYRINGE
1. Clean hands thoroughly with soap and water.
2. Write the name and hospital number of the patient on the tube in
which blood is to be collected. A printed label with these particulars can
also be used for patient identification.
3. Place the needle into the syringe. Keep the cap over the needle
capped till it is used. Check that the syringe works smoothly.
4. With the needle bevel up and parallel to the surface of the skin insert
it into the vein. Appearance of blood in the hub of the needle indicates
that the needle has successfully entered the vein. Release the tourniquet
as soon as blood enters the syringe.
5. Withdraw the piston slowly to avoid frothing.
6. After obtaining the requisite amount of blood, place a sterile gauze pad
over the point where the needle entered the skin and deftly withdraw the
needle simultaneously while applying pressure over the site.
7. Deliver the blood gently into the specified receiver. Cap it firmly to prevent
leakage.
8. Maintain light pressure on the gauze pad over venipuncture site till the
bleeding stops and then cover the puncture site with a small adhesive
dressing.
9. Destroy the needle in a special device (needle destroyer) immediately after
use. DONOT break, bend or recap needles after use.
10. Place the used swab, syringe and any other contaminated material in a
puncture resistant container for adequate disposal.
TYPES OF VACUUM TUBES
• Vacuum tubes with different colored caps are available. Each contains
a different anticoagulant and is used for various hematological tests as
described below
ARTERIAL BLOOD SAMPLE
• The artery is a less common site for blood collection. The most
common reason for taking blood from the artery is arterial blood gas
(ABG) analysis.
• The most common puncture site is the radial artery at the wrist and the
femoral artery in the groin. Blood can also be drawn from an arterial
catheter.
• In contrast to venous blood sampling, the withdrawal of a sample from
an artery requires training.
• COLLECTION OF CAPILLARY BLOOD
• Capillary blood is used for infants under 1 year of age and when it is not possible
to obtain venous blood.
• Common sites for drawing capillary blood are the heel and the ball of the middle
finger.
• Skin puncture is carried out with a needle or lancet. In adults and older children
blood can be obtained from a finger. The recommended site is the distal digit of
the third or fourth finger on its palmar surface, about 3-5 mm lateral from the nail
bed. Formerly, the earlobe was commonly used, but it is no longer recommended
because reduced blood flow renders it unrepresentative of the circulating blood.
• In infants, satisfactory samples can be obtained by a deep puncture of the plantar
surface of the heel in the area shown in figure. Because the heel should be very
warm, it may be necessary to bathe it in hot water.
• Capillary blood is free flowing, and thus it causes a greater risk of contamination
and disease transmission. Venous blood is therefore more convenient for handling
and it yields considerably more accurate results.
•Collection of Capillary Blood
1. Clean the area with 70% alcohol and allow to dry.
2. Puncture the skin to a depth of 2-3 mm with a sterile lancet.
3. Wipe away the first drop of blood with dry sterile gauze.
4. Collect the second and following drops onto a reagent strip or by a 10
ml or 20 ml micropipette.
• FINGER STICK SAMPLING
• The best locations for finger sticks are the center of the finger pads of the 3rd
(middle) and 4th (ring) fingers of the non-dominant hand. The tip of the finger or
the center of the finger should not be used. The sides of the finger where there is
less soft tissue should also be avoided.
ANTICOAGULANTS USED IN HEMATOLOGY
• The anticoagulants used commonly in the hematology laboratory are
• EDTA: Ethylene Diamine Tetra Acetic acid
• Sodium citrate
• Heparin
• Serum
• Serum is the part of the blood that contains neither blood cells nor
clotting factors.
• Serum is prepared by allowing the blood to naturally form a clot, and
then using a centrifuge to remove the blood cells and the clot. The
upper supernatant after centrifugation is the serum. To obtain serum
blood should be collected into a sterile tube that contains no
anticoagulants.
Ethylene-di-amine-tetra-acetic Acid (EDTA)
• Ethylenediaminetetraacetic Acid is a chelating agent produced as a
series of salts. The sodium and potassium salts of EDTA are powerful
anticoagulants, and they are especially suitable for routine
hematological work.
• Chemical Action
• EDTA acts by its chelating effect on the calcium molecules in the
blood. It removes calcium ions, which are essential for coagulation.
• Use
• EDTA is the anticoagulant of choice for blood counts and blood films.
It is also ideal for platelet counts as it prevents platelets from
clumping. This anticoagulant, however, is not suitable for coagulation
studies because it destroys clotting factors V and VIII.
Concentration
• The recommended concentration of EDTA is 1.5
± 0.25 mg/ml of blood. Excess of EDTA affects
both red cells and leukocytes causing shrinkage
and degenerative changes.
• A concentration exceeding 2 mg/ml of blood may
result in a significant decrease in packed cell
volume (PCV) and an increase in mean cell
Hemoglobin concentration (MCHC).
• The platelets are also affected; excess of EDTA
causes them to swell and then disintegrate,
leading to an artificially high platelet count.
• Therefore, it is important to ensure that the
correct amount of blood is added and that the
anticoagulant is thoroughly mixed in the blood
added to it.
• Trisodium citrate: This is used in coagulation studies in a ratio of 9
volumes of blood to one volume of anticoagulant (32g/L) (0.5ml
citrate+4.5ml blood). It binds calcium thus preventing coagulation. It
can also be used for estimation of ESR by Westergren method in a
ratio of 4 volumes of blood to 1 volume of sodium citrate.
• Heparin: The sodium or lithium salt of heparin is used at a
concentration of 10-20IU/ml of blood for osmotic fragility and for red
cell enzyme studies like glucose-6-phosphate dehydrogenase. It does
not change red cell size and is the best anticoagulant for osmotic
fragility. It can also be used for immunophenotyping.
• Heparin is not suitable for complete blood counts as it induces platelet
and leucocyte clumping. It should also not be used for making
peripheral smears as it gives a faint blue background color after
staining smears by Romanowsky dyes.
Trisodium Citrate
• Chemical Action
• Trisodium citrate removes free calcium ions by loosely
binding to them forming a calcium citrate complex.
• Use
• This anticoagulant is used for coagulation studies and
the estimation of the erythrocyte sedimentation rate
(ESR).
• Concentration
• For coagulation tests, nine volumes of blood are added
to one volume of the sodium citrate solution. For the
ESR, four volumes of blood are added to one volume of
the sodium citrate solution. Sodium citrate is available
in two concentrations: 3.2% and concentrations: 3.2%
and 3.8%.
Heparin
• Heparin is a natural substance which is
synthesized by the liver.
• Chemical Action
• Heparin neutralizes thrombin, an
essential clotting factor, with the aid of
a co-factor present in the albumin
fraction of plasma.
• Concentration
• The heparin concentration used is
10-20 international units (IU) per ml of
blood.
• Use
• Heparin is an effective anticoagulant and it does not alter the size of
red cells. It serves as a good dry anticoagulant when it's important to
minimize the chance of lysis after blood has been withdrawn.
• Heparin is the best anticoagulant for osmotic fragility and is suitable
for immunophenotyping.
• Heparinized blood is not recommended for cell counting because of its
clumping effect on platelets and leukocytes. It also should not be used
for making blood films because it gives a faint blue coloration to the
background when Romanowsky dyes are used.
• It inhibits enzyme activity, and it is not suitable for use in the study of
polymerase chain reaction (PCR) with restriction enzymes.
TYPES OF SAMPLES COLLECTED FOR
HEMATOLOGICAL INVESTIGATIONS
• Whole blood: This is used for performing complete blood counts
including reticulocyte count and for making peripheral blood films.
• Serum: If blood is allowed to clot at room temperature, a
straw-colored fluid appears which is called serum. This may be
admixed with red cells which can be removed by centrifugation at
1200g for 10 minutes. It is used for various biochemical tests and also
serum protein electrophoresis to diagnose plasma cell disorders such
as multiple myeloma. Serum lacks coagulation factors.
• Plasma: This is obtained by centrifugation of anticoagulated blood
and is used for coagulation studies.