CHAPTER TWO

BLOOD COLLECTION AND

ANTICOAGULANTS
2.1. Introduction to sample collection
 proper collection and reliable processing of blood
specimens is a vital part of the laboratory diagnostic
process in hematology as well as other laboratory
disciplines.
 Blood must be collected with care and adequate safety
precautions to ensure:
 test results are reliable
 contamination of the test sample is avoided and .
Introduction cont’d
 All material of human origin should be regarded as
potentially infectious
 When collecting blood sample, the operator should wear
disposable rubber gloves
 The operator is also strongly advised to cover any cuts,
abrasions or skin breaks on the hand with adhesive tape
before wearing gloves
Introduction cont’d
 Care must be taken when handling syringes and needles
 Do not recap used needles by hand
 needle-stick injuries are the most commonly encountered
accidents.
 Should a needle-stick injury occur:
 immediately remove gloves
 vigorously squeeze the wound while flushing the bleeding
with running tap water
 thoroughly scrub the wound with cotton balls soaked in
0.1% hypochlorite solution.
 Report to supervisor immediately
 Adhere to national guideline on HIV exposure risk
regarding post exposure prophylaxis (PEP)
Introduction cont’d
 Used disposable syringes , needles, other sharp items
such as lancets must be placed in puncture-resistant
container for subsequent decontamination or disposal.
2.2. Capillary blood collection
 Also referred to as microblood samples
collection/dermal puncture
 Iscollecting blood after an incision is made to
the skin with a lancet

 Preferred when only small quantities of

blood are required
 e.g.,
for hemoglobin quantitation, for WBC and
RBC counts and for blood smear preparation
 Capillary blood collection cont’d
 also used when venipuncture is impractical, e.g.,

 In infants

 In cases of severe burns

 In extreme obesity where locating the veins could be a problem

 In patient whose arm veins are being used for intravenous medication
 Capillary blood collection cont’d
 Sites of Puncture

 Adults and children:

 Palmer surface of the tip of the ring or middle finger or

free margin of the ear lobe

 Infants: plantar surface of the big toe or the heel.

Finger Prick
 Capillary blood collection cont’d

 The areas of the foot of a

baby or infant that are
suitable for obtaining capillary
blood
Heel Prick
 Capillary blood collection cont’d
Considerations:
 Edematous, congested and cyanotic sites should not
be punctured.
 Cold sites should not be punctured as samples
collected from cold sites give falsely high results of
hemoglobin and cell counts.
 Site should be massaged until it is warm
2.2.1. Capillary sample collection
method
 Materials Required
 Cotton or gauze pads
 70% alcohol or other skin
antiseptic
 sterile disposable lancet
(Automatic lancet)
 Capillary sample collection method cont’d
Technique:

1. Rub the site vigorously with cotton or a gauze pad moistened with 70%
alcohol
 to remove dirt and epithelial debris and
 to increase blood circulation in the area
 If the heel is to be punctured, it should first be warmed by immersion in a
warm water or applying a hot towel compress. Otherwise values
significantly higher than those in venous blood may be obtained.
 Cont’d

2. After the skin has dried, make a puncture 2-3mm deep with

a sterile lancet.

A rapid and firm puncture should be made with control of

the depth.

 A deep puncture is no more painful than a superficial

one and makes repeated punctures unnecessary.

Cont’d
 The first drop of blood which contains tissue juices

should be wiped away

 The site should not be squeezed or pressed to get

blood since this dilutes it with fluid from the tissues.

 a freely flowing blood should be taken or a moderate

pressure some distance above the puncture site is

allowable
 Capillary blood collection cont’d

3. Stop the blood flow by applying slight pressure at the site preferably with

a gauze pad

 Cotton balls are not recommended, because they stick to the site

and can initiate bleeding

 Advantages of Capillary Blood

 obtained with ease.

 the preferred specimen for making peripheral blood films since no

anticoagulant is added that may affect cell morphology.

 Cont’d
 Disadvantages of Capillary Blood
 only small amounts of blood can be obtained and repeated
examinations require a new specimen.
 Platelet count cannot be performed on capillary blood
 some platelets are unavoidably lost by adherence onto the wound.
 Precision is poorer in capillary than venous blood
 because of variation in blood flow and dilution with interstitial fluid.
 Blood in microtubes frequently hemolyse
 hemolysis interferes with most laboratory tests.
2.3. Venous Blood Collection
 Collection of blood from the vein (venipuncture)
 Also referred to as phlebotomy
 Necessary for most tests that require anticoagulation or
larger quantities of blood, plasma or serum.
 Sites of Puncture
 Veins of the forearm are preferred; wrist or ankle can
also be used
 veins in the antecubital fossa of the arm are the
preferred sites
 they are larger than those in the wrist or ankle

regions
 hence are easily located and palpated in most

people.
 -The three main veins in the forearm
1. Medial Cubital
 First choice well
anchored and easy to
penetrate
2. Cephalic
 On the outside surface
 Well anchored

3. Basilic
 Not well anchored, tends
to roll, painful and can
cause nerve damage
Anterior surface of the left arm showing
veins most suitable for venepuncture.
 Venous Blood Collection cont’d
Venous blood collection in infants and children
 presents special problems
 small size of the veins
 difficulty in controlling the patient; needs excellent interpersonal skill
 Require experience
 Areas also used for obtaining blood
 external jugular vein in the neck region
Venous Blood Collection cont’d
 Materials:
 Gloves
 vacutainer tube
 vacutainer tube holder and Two-way needle
 sterile syringe and needle (if the syringe method is used)
 tourniquet
 gauze pads or cotton,
 70% alcohol or suitable skin antiseptic
 test tubes with or without anticoagulant (for syringe method)
 Sharp container
 Band Aid (to stop further bleeding)
Vacutainer Tubes color
codes??

Vacutainer tube holder

Needles

 Sterile, Disposal and

for Single use only
 Color coded for size or
gauge
 Hollow stainless steel
shafts
 Bevel or slant at the
end
Needle Features

 Bevel or slant:
 Must face up
 Shaft:
 cylindrical portion, length
ranges from .65 to 3.80 cm
 Hub:
 attaches the needle to the
holder
 Gauge:
 The size of the needle, refers
to the diameter of the needle
Needle
 Needle Size or Gauge
depends on the size and
depth of the vein to be
punctured
 Gauge is the diameter of the
needle
 The larger the gauge number
the smaller the needle
 Range from 16 – 25
 Routine: 21 gauge
 Children: 23 gauge
 Most common used 21g by
3.80 cm
 Colored coded for size
Safety Needles

 Allows the user’s hand

to remain behind the
needle
 Always use a safety
needle when available
 Minimizing exposure
to blood borne
pathogens
Tourniquet

 Slow down venous flow

without affecting arterial
flow
 To enlarge the vein for
easier location and easier
penetration
 To assess the width,
direction and depth of
veins
 Should be left on for no
longer than one minute
Gloves

 ‘Universal Precautions”
require the wearing of gloves
 Gloves provide protection
from infection
 A new pair of gloves must be
worn for each client and for
each new procedure
Sharp container
 2.3.1. Vacutainer technique of venous blood
collection
 Techniques

1. Assemble the necessary materials and equipment

2. thread the short end of the double-pointed needle

into the holder and push the tube forward until the
top of the stopper meets the guide mark on the
holder.
 Vacutainer technique cont’d
Needle Holder and Adapter
 The second pointed
needle with rubber sleeve
resides in the holder
 Push the tube directly into
the middle of the holder
for proper collection
 Second needle will
penetrate top of tube
 Vacutainer technique cont’d
 The point of the needle will thus be embedded
in the stopper without puncturing it and loosing
the vacuum in the tube.

3. Identify the right patient and allow him/her to

sit comfortably preferably in an armchair
stretching his/her arm.

4. Reassure the patient

5. Apply the tourniquet

6. Prepare the arm by swabbing the antecubital

fossa with a gauze pad or cotton moistened
with 70% alcohol.
 Vacutainer technique cont’d
Tourniquet Application
 Position the tourniquet
7.5 – 10 cm above the
vein puncture site with
strip equal on both
sides
 Pull Left side over the
Right side and make
an X
 Tourniquet Application cont’d

 With X in place, tuck a

loop
 Have both ends of
strip facing up
 End of the tourniquet
that is facing up is
used for easy removal
 Cleaning the Arm
 Clean the arm using 70%
isopropyl alcohol
 Clean area in concentric
circles starting at site and
ending outside of site. Do
not re-touch the cleaned
area
 Let air dry
 Vacutainer technique cont’d

7. Grasp the back of the patient’s arm at the elbow and anchor the

selected vein by drawing the skin slightly taut over the vein

8. insert the needle properly into the vein;

 the index finger is placed along side of the hub of the needle with the

bevel facing up

 The needle should be pointing in the same direction as the vein.

9. Then the point of the needle is advanced 0.5-1.0cm into the

subcutaneous tissue (at an angle of 450) and is pushed forward at a

lesser angle to pierce the vein wall.

 Vacutainer technique cont’d
10. When the needle is properly in the vein, the vacuum tube is pushed
into the needle holder all the way so that the blood flows into the
tube under vacuum.

11. The tourniquet should be released the moment blood starts

entering the vacuum tube
 otherwise, some hemoconcentration will develop after one
minute of venous stasis.

12. After drawing the required blood sample, apply a ball of cotton to
the puncture site and gently withdraw the needle.

13. Instruct the patient to press on the cotton

 Vacutainer technique cont’d
14. Remove the tube from the vacutainer holder and if the tube is with
anticoagulant, gently invert several times
 Invert 8-10 times for EDTA tube

15. Label the tubes with patient’s name, hospital number and other
information required by the hospital (before the patient leaves the
collection area)

16. Re-inspect the venipuncture site to ascertain that the bleeding has
stopped.

17. Do not let the patient go until the bleeding stops

 If bleeding does not stop apply band aid

18. Bid farewell to the patient with smile

Inverting and Mixing the Tubes

 Tubes with additives

must be gently mixed
 Gently invert several
times for proper
mixing
 Advantages of the Vacutainer Method of Venous Blood
Collection:

 an ideal means of collecting multiple samples with ease

 eliminates many of the factors that cause hemolysis.
 no preparation of anticoagulants and containers needed.
 One can choose among a wide range of tube size with or
with out anticoagulant.
 Because the evacuated tubes are sterile, avoids possible
bacterial contamination
 Thus, an ideal blood sample for microbiological analysis
 Anticoagulants
 EDTA
ethylenediaminetetraaceti
c acid in a tripotassium or
disodium base (Purple):
prevents clotting by
binding Ca.
 Used in Heamatology or
TBC and CD4
 Anticoagulants
 Sodium Citrate
(Light Blue):
prevents clotting by
binding the calcium.
 Used for
coagulation workup
(PT and APTT)
 Anticoagulants
 Heparin (Green): Three
types: ammonium, lithium,
and sodium. Prevents
clotting by inhibiting
thrombin. Used for plasma
chemistry testing
 Use appropriate type of
heparin
 Anticoagulant/Additive
 ANTICOAGULANT
 Potassium or ammonium
oxalate (Gray): prevents clotting
by binding the calcium. Used in
glucose testing

ADDITIVE
 Sodium Fluoride (Gray): inhibits
the metabolism of glucose by
the cells. Used for glucose
tolerance testing
Additives

 Clot activator:
 initiate or enhance
coagulation. Can be
thrombin, glass or silica
particles
 Gel Separator:
 forms a barrier between the
cells and the serum portion
after the blood has been
spun
 Used for Chemistry Testing
Tube Stoppers

 Red/Glass: no additives
 Red/Plastic: clot activator
 Red/Gray or marbled: gel separator
 Green: Heparin (three types)
 Mint Green: Heparin with gel separator
 Lavender: EDTA
 Light Blue: Sodium citrate
 Gray: potassium oxalate and sodium fluoride
 Hemogard: plastic stopper with rubber inside.
Used for safety
Colors of Tubes
2.3.2. Syringe method of blood
collection
 Remove the syringe from its protective wrapper and the
needle from the cap
 assemble them allowing the cap to remain covering the
needle until use.
 Attach the needle so that the bevel faces in the same
direction as the graduation mark on the syringe
 Check to make sure:
 the needle is sharp
 the syringe moves smoothly
 there is no air left in the barrel
Syringe method cont’d

 Identify the right patient and allow him/her to sit

comfortably preferably in an armchair stretching his/her
arm.
 Reassure the patient
 Apply the tourniquet
 Prepare the arm by swabbing the antecubital fossa with
a gauze pad or cotton moistened with 70% alcohol.
 Grasp the back of the patient’s arm at the elbow and
anchor the selected vein by drawing the skin slightly taut
over the vein
 Syringe method cont’d
 Using the assembled syringe and needle, enter the skin first
and then the vein
 insert the needle properly into the vein;
 the index finger is placed along side of the hub of the
needle with the bevel facing up
 The needle should be pointing in the same direction as
the vein.
 The plunger is drown back to create suction pressure to
draw the blood
Venipuncture technique using
needle and syringe
 Advantages of Venous Blood:
 various tests to be repeated in case of accident or breakage
 checking of a doubtful result
 performance of additional tests
 aliquots of the specimen (plasma and serum) may be frozen
for future reference.
 reduces the possibility of errors resulting from
 tissue dilution with interstitial fluid
 constriction of skin vessels by cold that may occur in
taking blood by skin puncture.
 Disadvantages of Venous Blood:

 Lengthy procedure
 technically difficult in children, obese individuals and in
patients in shock.
 more likelihood of occurrence of Hemolysis
 hemolysis must be prevented because it leads to
lowered red cell counts and interferes with many
chemical tests.
 Hematoma (or blood clot formation inside or outside the
veins) may also occur
Adult Venipuncture Steps

1. PREPARE PAPERWORK
2. IDENTIFY YOUR SELF AND THE CLIENT
3. VERIFY DIET RESTRICTIONS
4. ASSEMBLE EQUIPMENT
5. WASH HANDS AND PUT ON GLOVES
6. REASSURE AND POSITION CLIENT
Adult Venipuncture Steps,
cont’d
7. APPLY TOURNIQUET
8. SELECT VENIPUNCTURE SITE
9. CLEANSE SITE
10. PICK UP NEEDLE AND REMOVE COVER AND
INSPECT
11. ANCHOR VEIN WITH THUMB
12. INSERT THE NEEDLE INTO THE VEIN AT A 15
– 30 DEGREE ANGLE WITH THE BEVEL
FACED UP
Procedure
Adult Venipuncture Steps,
cont’d
13. FILL TUBES IN ORDER OF DRAW, MIX TUBES
WELL, RELEASE THE TOURNIQUET AFTER
THE FIRST TUBE IS FILLED, WITHDRAW LAST
TUBE FROM HOLDER
14. WITHDRAW NEEDLE
15. DISPOSE OF NEEDLE
16. LABEL TUBES
17. CHECK CLIENT'S ARM AND APPLY BANDAGE
Procedure
Procedure
Adult Venipuncture Steps,
cont’d

18. DISPOSE OF CONTAMINATED MATERIALS

19. THANK CLIENT
20. REMOVE GLOVES, WASH HANDS
Complications Associated
with Blood Collection

 Avoid scars and burns

 Edema:
 Abnormal accumulation of fluid in the tissues. Lab
results will be inaccurate
Complications Associated
with Blood Collection
 Hematoma: is a
swelling or mass of
blood under the skin.
This area is usually
sore and painful.
-Should be avoided
Complications Associated
with Blood Collection
 Obesity:
 Veins may be deep. The cephalic is the vein of
choice
 Damaged veins:
 Feel hard and cord like, difficult to penetrate and
should be avoided
Complications Affecting the
Client
 Allergies:
 May have a reaction to the alcohol or band-aid
 Seizures:
 Remove the needle immediately
 Hold pressure to the site without restriction of the
client’s movements
 Excessive Bleeding:
 Pressure should be maintained for more than 5
minutes
Complications Affecting the
Client
 Fainting:
 If there is history, lie the client down.
 If the client faints during the procedure, remove the
needle immediately.
 Put the client’s head between their knees and make
them take deep breaths.
 Hematoma:
 Ifone starts to form during the procedure, remove
needle and apply pressure
Complications Affecting the
Client
 Nausea:
 client should be made comfortable, deep breaths
 Infection:
 Rare occurrence, clean site properly and apply a band-aid
 Pain:
 A little pain is associated with the procedure
 No blind probing
 Petechiae:
 Red spots under the skin which appear when a tourniquet is left
on.
 Usually there is capillary fragility
Collection Techniques
Affecting the Specimen
 Hemoconcentration:
 Prolonged application of the tourniquet, vigorous and probing
 Hemolysis: results from the destruction of the red cells
 shaking the tube hard
 drawing from a hemtoma
 pulling on a syringe to quickly
 probing
 to small a needle
 forcing the blood into another tube
Collection Techniques
Affecting the Specimen
 Partially filled tubes:
 Filling additive tubes until the vacuum is exhausted is
important for proper ratio of anticoagulant to blood
 Very important for coagulation studies, under
filled tubes are not acceptable
 Specimen Contamination:
 Alcohol can cause contamination, touching the site,
not following order of draw
Safety Reminders

 Always use standard or universal blood

precautions when drawing blood
 Always wear gloves when drawing blood
 Identify patient by asking for a name or check
armband
 Do not allow tourniquet to remain on the arm for
more than one minute
 Properly label tubes after the blood is in them