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A. Blood Collection Tubes

 Gold - Clot activator and gel for
serum separation. Used for
serum determinations in
chemistry and for routine blood
donor screening and diagnostic
testing of serum for infectious
 Light Green - Lithium heparin
and gel for plasma separation.
Used for plasma determinations
in chemistry.
 Red - Silicone coated (glass),
Clot activator, Silicone coated
(plastic). Used for serum
determinations in chemistry
and for routine blood donor
screening and diagnostic testing
of serum for infectious disease.
 Orange - Thrombin-based clot
activator with gel for serum
separation. Used for stat serum
determinations in chemistry.
 Royal Blue - Clot activator
(plastic serum), K2EDTA
(plastic). Used for trace-
element, toxicology, and
 Green - Sodium heparin or
Lithium heparin. Used for
plasma determinations in
 Gray - Potassium
oxalate/sodium fluoride, Sodium
fluoride/Na2EDTA, Sodium
fluoride (serum tube). Used for
glucose determinations. Oxalate
and EDTA anticoagulants will
give plasma samples. Sodium
fluoride is the antiglycolytic
 Tan - K2EDTA (plastic). Used
for lead determinations. This
tube is certified to contain less
than .01 μg/mL(ppm) lead.
 Lavender - Liquid K3EDTA
(glass), Spray-coated K2EDTA
(plastic). K2EDTA and K3EDTA
for whole blood hematology
determinations. K2EDTA may
be used for routine
immunohematology testing, and
blood donor screening.
 White - K2EDTA and gel for
plasma separation. used in
molecular diagnostic test
methods (such as, but not
limited to, polymerase chain
reaction [PCR] and/or branched
DNA [bDNA] amplification
 Pink - Spray-coated K2EDTA
(plastic). used for whole blood
hematology determinations.
May be used for routine
immunohematology testing and
blood donor screening.
 Light Blue - Buffered sodium
citrate 0.105 M (≈3.2%)
glass0.109 M (3.2%) plastic.
Citrate, theophylline,
adenosine, dipyridamole
 Clear – None (plastic). Used as
a discard tube or secondary
specimen tube

B. Complete Venipuncture
and Skin Puncture
Equipment and their
uses or functions.

I. Venipuncture

 Tourniquet – used to provide a
barrier against venous blood
flow to help locate a vein. It
must not be applied 2-4 inches
above puncture site left no
longer than 1 minute prior to
drawing of blood.
 Collection tubes – evacuated
tube system is commonly used.
Plastic tubes are recommended,
while glass tubes are coated
with silicone inside to prevent
hemolysis and cell adherence.
 Needles – in selecting needle
gauge for phlebotomy always
use the appropriate size for the
vein diameter to prevent vein
collapse and hemolysis.
Needles are made to be screwed
into the evacuated tube holder
or to be attached to the tips of
syringes. The needle gauges for
drawing blood range from 20 to
25 gauge. The most common
needle size for adult
venipuncture is 21 gauge with
a length of 1 inch.
 Needle holder – prevents the
possibility of needle sticks.
They are disposable and must
be discarded after

single use with the needle
 Winged infusion sets (butterfly)
– an intravenous device with
short needle and a thin tube
with attached plastic wings.
Useful in difficult blood draws
especially in children and
difficult veins.
 Syringes – useful in drawing
blood from pediatric, geriatric
or other patients with tiny,
fragile or “rolling” veins that
would not be able to withstand
vacuum pressure from
evacuated tubes.
 Solutions for Skin preparation
– 70% isopropyl alcohol is
commonly used. It is used to
clean the site.

II. Skin Puncture

 Capillary tubes – with or
without heparin added.
 Microcollection tubes –
available with or without
additives, and the cap colors on
the tubes correspond with the
colors on vacuum tubes.

C. Collection sites for
Venipuncture and Skin
Puncture (both for adults
and children)

I. Venipuncture

 Median cubital vein is the first
 Cephalic vein – most prominent
in obese patients Basilic veins
can also be used.

II. Capillary Puncutre

 Heel or finger can be used.
 In infants, only the heel is used
– the lateral or medial surface
of the plantar side of the heel.
 In older children and adult –
the palmar surface of the distal
portion of the third and fourth
finger on the non-dominant
hand may be used.

D. Complications
encountered during blood

 Ecchymosis (Bruise) – caused
by leakage of a small amount of
fluid around the tissue.
 Syncope (Fainting) – it is the
second most common
complication. Remove the
needle immediately if the
patient starts to faint.
 Hematoma – a leakage of a
large amount of fluid around
the puncture site causes the
area to swell. If the area starts
to swell, remove the needle and
apply pressure for several
 Failure to draw blood – the
major reason is the vein is
missed, often because of
improper needle positioning.
The needle should be inserted
completely into the vein with
the slanted side (bevel) up, at
an angle of 15 to 30 degrees.
Other reasons are device
malfunction and vacuum loss in
evacuated tubes.
 Petechiae – small red spots
indicating small amounts of
blood that have escaped into
the epithelium. It indicates a
coagulation problem and
should alert the phlebotomist
for prolonged bleeding.
 Edema – it is an abnormal
accumulation of fluid in the
intercellular spaces of the
tissues. Edematous sites should
be avoided for venipuncture.
 Obesity – the cephalic vein is
often the only vein that can be
felt in obese patients. When
palpating do not depress the
area so much because you
might miss the vein suspended
in the fatty tissue. Usually the
error in palpating obese
patients is thinking that the
vein is buried deep below fat,
this is not always the case.
 Intravenous therapy – drawing
blood from an arm with IV
catheter should be avoided if
possible. If not possible, apply
tourniquet below the IV site
and draw blood below the IV
site. Have the nurse stop the
infusion 2 minutes before
drawing blood. Discard the first
5mL of blood collected before
obtaining samples for testing.
Note: the problem with this is
that the IV catheter might be
dislodged, as much as possible
find another site.
 Hemoconcentration – increased
concentration of larger
molecules and analytes in the
blood as a result of a shift in
water balance. Caused by
leaving the tourniquet on the
arm for too long. It should not
take more than 1 minute, if the
vein is not located within 1
minute it should be removed
for 2 minutes and reapplied
before the venipuncture.
 Hemolysis – can cause the
plasma or serum to appear
pink or red due to the rupture
of RBCs with the consequent
escape of hemoglobin. Can
occur if small gauge needle is
used in difficult draws; pulling
back the plunger too quickly;
forces blood from syringe into a
tube; shakes the tube; or if the
sample is contaminated with
alcohol or water
 Burned, Damaged, Scarred and
Occluded Veins – should be
avoided because blood does not
flow freely in these sites.
 Seizures and tremors – remove
needle immediately to prevent
injury. Preventing injury from
nearby objects should ensure
the patient’s safety.
 Vomiting and choking – the
patient’s head is positioned so
that he/she does not aspirate
any vomit. Keep the patient
from hitting his/her head.
 Allergies – some patients may
be allergic to skin antiseptic
substances other than alcohol,
adhesive bandages and tape.
Sensitivity to latex should be
 Mastectomy patients – requires
physician permission before
blood is drawn from the same
side as a prior mastectomy
even in the case of bilateral
mastectomies. The pressure on
the arm with the tourniquet
can lead to pain or
 Inability to Obtain a Blood
Specimen – if two unsuccessful
attempts at collection have
been made, it is recommended
that the phlebotomist seek the
assistance of another caregiver
with blood collection expertise.
The patient has the right to
refuse to give a blood specimen.

E. Venipuncture Procedure
in Adults.

1. ID the patient and look for the
doctor’s order.
2. Wash hands, wear gloves and
choose a comfortable place to
set up the biological sampling
3. Lay out a clean disposable mat
with all the equipment
necessary to collect blood
4. Examine both arms to find the
best vein. Locate the puncture
site; apply the tourniquet(less
than 2 minutes).
5. Cleanse the area with an
alcohol wipe. Wipe the area in
a circular motion making sure
the area is thoroughly cleaned.
6. If it is necessary to feel the vein
again, do so, but cleanse the
area again with an alcohol
7. Fix the vein by pressing down
on the vein about 1 inch below
the proposed point of entry and
pull the skin taut.
8. Remove the needle shield.
9. Approach the vein in the same
direction the vein is running,
holding the needle so that it is
at an approximately 15° angle
with the participant's arm.
10. Push the needle, with bevel
facing up, firmly and
deliberately into the vein, If the
needle is in the vein, blood will
enter the syringe and you can
start pulling the plunger
11. For collection, loosen the
tourniquet immediately after
blood flow is established.
12. When you have the desired
volume of blood, slowly
removed the syringe and apply
the dry gauze.
13. Ask the patient to apply
pressure for 10 minutes.
14. Properly label the specimen.
15. Dispose the used material
properly (if possible cut the
needle using a needle cutter to
avoid accidental prick).

F. Venipuncture Procedure
in Children and Infants.

1. Wash hands and place on
2. Identify the patient.
3. Prepare and organize
venipuncture equipment. A
23g butterfly attached to a
pediatric vacutainer holder.
Pediatric vacuum tubes can be
used on prominent veins. For
smaller veins use a 23g
butterfly attached to a 3-5 ml
4. Restrain the child if necessary
according to the
recommendations (roll the
infant in a blanket exposing
only the puncture site).
5. Place a pediatric size
tourniquet around the bicep
muscle in the upper arm.
Palpate for a vein in the
antecubital fossa.
6. Once the vein has been located
cleanse site in concentric circles
with 70% isopropyl alcohol. Let
air dry.
7. Anchor the vein by pulling skin
down with thumb or index
8. Make sure to keep needle out of
sight from child. Enter vein
with butterfly needle at a 15 to
30 degree angle. Check for
“flash” of blood in the tubing. If
no blood appears, gently
redirect until “flash” appears.
As soon as blood appears in the
tubing of the butterfly, pull the
syringe plunger to fill with
blood (butterfly/syringe
method) or engage vacuum
tubes into holder (vacutainer
method) and fill to capacity.
Make sure not to exceed
maximum daily blood volume.
(See volume chart) After
collection is completed activate
the safety mechanism on the
Butterfly device. Hold direct
pressure on the puncture site
with 2 x 2 gauze sponges until
bleeding has stopped. Apply a
clean 2 x 2 gauze sponge folded
into fourths and apply paper
9. Discard the entire assembly
device in an approved sharps
disposal container.
10. Gently invert additive tubes
and label tubes with:
a. Patient’s name b. Medical
record number c. Date
d. Time of draw e. Blood
collector’s initials and notify
the infant’s nurse of the total
blood volume collected, or
record in appropriate log.

G. Skin Puncture in Adults.

1. Gloves must be worn when
performing skin punctures.
2. ID the patient.
3. Prepare the puncture
4. If finger is cold, wrap in warm
compress at a temperature no
higher than 42° for three to five
minutes to warm site.
5. Briskly rubbing the heel or
fingertip will improve
6. Cleanse the area with 70%
alcohol pad and let it air dry.
7. Place the puncture device on
the patient's skin and activate
the release mechanism.
8. Wipe the first drop of blood
away with dry gauze
(contaminated with tissue
9. Collect blood using capillary
tube action (never put too much
pressure just to force the blood
10. Label the specimen and dispose
the materials properly.

H. Skin Puncture in
Children and infants.

1. Check the computer label or lab
requisition to see what test is
ordered. Obtain and select
appropriate equipment for the
heel puncture procedure.
2. Properly identify the infant by
matching the computer label
with the infant’s identification
number located on the bracelet.
3. Place gloves on hands.
4. Infant should be positioned in
the supine position (face up).
Allow the foot to hang lower
than the torso to improve blood
5. Activate and place a chemical
heel warmer on the infant’s
heel for 3 to 5 minutes. This
will increase the blood flow to
the area by 7 fold.
6. Assemble all of your
equipment. Do not lay any
equipment in bassinet. Use
shelf underneath bed or top of
7. After stabilizing the infant’s
foot, cleanse the foot with a
70% alcohol pad and dry with a
piece of sterile gauze. Alcohol
residue on the infant’s foot may
cause a stinging sensation and
hemolysis of the red cells.
8. Place your thumb on the inside
bottom of the foot and push the
skin across the heel toward the
outside of the heel.
9. Puncture the most medial
section (outside) of the plantar
surface of the heel. Wipe the
first drop of blood with a piece
of gauze since this blood has
been diluted with tissue fluid
and may contaminate the
10. Draw blood by capillary tube
action; mix well with the
anticoagulant (red band).
Dispose properly the materials.

I. Specimen Handling and
Quality Assurance in
Specimen Collection.

 Technical competence

A phlebotomist should be well
trained in all phases of blood
collection. Continuing
education is encouraged.

 Collection Procedures

Procedures must be reviewed.
Patient preparation and
identification are important.

 Anticoagulants and

The kind of anticoagulant used
must be suitable for the test.
Proper mixing should be
followed following
manufacturer’s instructions.
Always check the validity and
quality of tubes to be used.

 Requirements for a Quality

 Patient properly identified
 Patient properly prepared for
 Specimens collected in correct
order and labeled correctly
 Correct anticoagulants and
preservatives used
 Specimens properly mixed
 Specimens not hemolyzed
 Specimens requiring fasting
are collected on time
 Timed specimen drawn at the
correct time

 Blood collection attempts

A person may attempt to draw
blood only twice. If
unsuccessful, another
individual must be called to do
the job. Inform the
physician/nurse if no blood was

 Collection of specimens for
blood culture

The rate of contamination for
blood culture must be less than
3%. 2-3 blood collections are
required depending on the case
and must not be collected on
the same site. The 2 remaining
samples on the next day will
follow the first sample taken.
Blood volume for adults: 10-20
mL; Infants: 1-5mL.

 Quality control and preventive
maintenance on specimen
collection instruments:

1. Periodic maintenance should
be followed on all
instruments using the
manufacturer’s advice.
2. The service report on all
repairs and periodic
maintenance should be kept
and filed.

 Reasons for specimen rejection:

a. The test request and tube
identification do not match
b. The tube is unlabeled.
c. Specimen is hemolyzed
d. Wrong collection time
e. Wrong tube/ anticoagulant for
the specimen/test.
f. Clotted specimen for whole
blood examinations
g. Lipemic specimen – case to
case basis
h. Contaminated with IV fluid

 Specimen handling

Preanalytical phase – the pre
testing period. Includes the test
requisition and all specimens
handling before testing. Each
test has their own specimen
handling requirements and
must be observed carefully.


A. Making of a Peripheral
Blood Smear

1. Draw blood from a patient.
2. Blood from the syringe or the
blood transferred on a red top
tube can be used to make
3. Place a very small drop of blood
near the end of the sample
4. Place the end of the spreader
slide on the sample slide so
that the short sided edge of the
spreader is just below the drop
of blood (Holding the spreader
at an angle of 30o)
5. Quickly drag the spreader
along the entire length of the
sample slide in one fluid
motion. (Factors to be
considered: Size of blood drop,
angle, speed, etc.)

B. Differential Count

1. Make a peripheral blood smear.
2. Stain using the smear using the
Wright’s stain.
3. Examine under the microscope.
4. Identify the different white blood
5. Record the results.

C. Hematocrit and Hemoglobin

1. Perform a skin puncture procedure.
2. Seal your capillary tube (red banc)
with clay and wax.
3. Centrifuge the tubes.
4. Compare it on the chart for
hematocrit and hemoglobin.
5. Report your results (use decimal or
percentage form when reporting for


A. Veins of the forearm

B. Proper and Improper Needle Insertion

C. Areas for Skin Puncture

D. How to Assemble and Use a Multi-sample Needle

Hematology 1

January 9, 2014

Submitted by: Al Glen D. Egarle
Mohammad Hattah C. Macala

Submitted to: Ma’am Jean Bedia