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Phlebotomy Manual

Prepared by,

Atiq Ur Rehman

DMLT

BS MLT

Riphah International University Lahore Campus

03027557000

03137557000
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BLOOD COLLECTION:

A phlebotomist is a trained Health professional specially drawing the blood from patients
for testing.

Objectives:

• Describe and perform the venipuncture process including:

1. Appropriate clothing and protective equipment
2. Ensuring the comfort of the patient
3. Proper patient identification procedures.
4. Proper equipment selection and use.
5. Proper labeling procedures and completion of laboratory requisitions.
6. Order of draw for multiple tube phlebotomy.
7. Preferred venous access sites, and factors to consider in site selection, and ability
to differentiate between the feel of a vein, tendon and artery.
8. Patient care following completion of venipuncture.
9. Safety and infection control procedures.
10. Quality assurance issues.
• Identify the additive, additive function, volume, and specimen considerations to be
followed for each of the various color coded tubes.
• Summarize the problems that may be encountered in accessing a vein, including the
procedure to follow when a specimen is not obtained.

VENIPUNCTURE PROCEDURE

The venipuncture procedure is complex, requiring both knowledge and skill to perform.
Each phlebotomist generally establishes a routine that is comfortable for her or him.

Phlebotomists are considered to have occupational exposure to blood borne pathogens.

The performance of routine vascular access procedures by skilled phlebotomists requires,
at a minimum, the use of gloves to prevent contact with blood. Laboratory coats or work
needle are not typically needed as PPEs during routine venipuncture,. It is an employer's
responsibility to provide, clean, repair, replace, and/or dispose of personal protective
equipment/clothing. As part of presenting a professional appearance, an institutional
dress code may include wearing of a laboratory coat.
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Several essential steps are required for every successful collection procedure:

i. Patient relaxation is important and has the patient been seated for at least 5 minutes to
avoid being rushed or confused?
ii. Carry out hand hygiene before and after each patient procedure, before putting on and
after removing gloves.
iii. Identify the patient using two different identifiers, asking open ended questions such as,
"What is your name?" and "What is your date of birth?"
iv. Assess the patient's physical disposition (i.e. diet, exercise, stress, basal state).
v. Check the requisition form for requested tests, patient information, and any special
requirements.
vi. Label the collection tubes at the bedside or drawing area.
vii. Select a suitable site for venipuncture.
viii. Prepare the equipment, the patient and the puncture site.
ix. Perform the venipuncture, collecting the sample(s) in the appropriate container(s).
x. Recognize complications associated with the phlebotomy procedure.
xi. Assess the need for sample recollection and/or rejection.
xii. Promptly send the specimens with the requisition to the laboratory.

REQUEST FORM

A request form must accompany each sample submitted to the laboratory. This request form
must contain the proper information in order to process the specimen. The essential elements of
the request form are:

• Patient's surname, first name, and middle initial.

• Patient's ID number.
• Patient's date of birth and sex.
• Requesting physician's complete name.
• Source of specimen. This information must be given when requesting microbiology,
cytology, fluid analysis, or other testing where analysis and reporting is site specific.
• Date and time of collection.
• Indicating the test(s) requested.

An example of a simple requisition form with the essential elements is shown below
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LABELING THE SAMPLE

A properly labeled sample is essential so that the results of the test match the patient.
The key elements in labeling are:

• Patient's surname, first and middle.

• Patient's ID number.
• NOTE: Both of the above MUST match the same on the request form.
• Date, time and initials of the phlebotomist must be on the label of EACH tube.

Automated systems may include labels with bar codes.

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EQUIPMENT:

THE FOLLOWING ARE NEEDED FOR ROUTINE VENIPUNCTURE:

• Evacuated Collection Tubes

The tubes are designed to fill with a predetermined volume of blood by vacuum. The
rubber stoppers are color coded according to the additive that the tube contains. Various
sizes are available. Blood should NEVER be poured from one tube to another since the
tubes can have different additives or coatings.

• Needles

The gauge number indicates the bore size: the larger the gauge number, the smaller the
needle bore. Needles are available for evacuated systems and for use with a syringe,
single draw or butterfly system.

• Holder/Adapter

Use with the evacuated collection system.

• Tourniquet

Wipe off with alcohol and replace frequently.

• Alcohol Swabs

70% isopropyl alcohol.

• Povidone-iodine wipes/swabs

Used if blood culture is to be drawn.

• Gauze sponges

For application on the site from which the needle is withdrawn.

• Adhesive bandages / tape

Protects the venipuncture site after collection.

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• Needle disposal unit

Needles should NEVER be broken, bent, or recapped. Needles should be placed in a proper
disposal unit IMMEDIATELY after their use.

• Gloves

Can be made of latex, rubber, vinyl, etc.; worn to protect the patient and the phlebotomist.

• Syringes

Maybe used in place of the evacuated collection tube for special circumstances.

ORDER OF DRAW

Blood collection tubes must be drawn in a specific order to avoid cross-contamination of

additives between tubes. The recommended order of draw for plastic or glass collection
tubes is:

I. First - blood culture bottle or tube (yellow or yellow-black top)

II. Second - coagulation tube (light blue top). If just a routine coagulation assay is the only
test ordered, then a single light blue top tube may be drawn. If there is a concern
regarding contamination by tissue fluids or thromboplastins, then one may draw a non-
additive tube first, and then the light blue top tube.
III. Third - non-additive tube (red top)
IV. Last draw - additive tubes in this order:
a. SST (red-gray or gold top). Contains a gel separator and clot activator.
b. Sodium heparin (dark green top)
c. PST (light green top). Contains lithium heparin anticoagulant and a gel separator.
d. EDTA (lavender top)
e. ACDA or ACDB (pale yellow top). Contains acid citrate dextrose.
f. Oxalate/fluoride (light gray top)

NOTE: Tubes with additives must be thoroughly mixed. False test results may be
obtained when the blood is not thoroughly mixed with the additive.
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PHLEBOTOMY PROCEDURE ILLUSTRATED:

❖ Patient identification
❖ Filling out the requisition
❖ Equipment
❖ Apply tourniquet and palpate for vein
❖ Sterilize the site
❖ Insert needle
❖ Drawing the specimen
❖ Drawing the specimen
❖ Releasing the tourniquet
❖ Applying pressure over the vein
❖ Applying bandage
❖ Disposing needle into sharps
❖ labeling the specimens

VENIPUNCTURE SITE SELECTION:

Although the larger and fuller median cubital and cephalic veins of the arm are used
most frequently, the basilic vein on the dorsum of the arm or dorsal hand veins are also
acceptable for venipuncture. Foot veins are a last resort because of the higher
probability of complications.

Certain areas are to be avoided when choosing a site:

• Extensive scars from burns and surgery - it is difficult to puncture the scar tissue
and obtain a specimen.
• The upper extremity on the side of a previous mastectomy - test results may be
affected because of lymphedema.
• Hematoma - may cause erroneous test results. If another site is not available,
collect the specimen distal to the hematoma.
• Intravenous therapy (IV) / blood transfusions - fluid may dilute the specimen, so
collect from the opposite arm if possible. Otherwise, satisfactory samples may be
drawn below the IV by following these procedures:
o Turn off the IV for at least 2 minutes before venipuncture.
o Apply the tourniquet below the IV site. Select a vein other than the one
with the IV.
o Perform the venipuncture. Draw 5 ml of blood and discard before drawing
the specimen tubes for testing.
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• Lines - Drawing from an intravenous line may avoid a difficult venipuncture, but
introduces problems. The line must be flushed first. When using a syringe inserted
into the line, blood must be withdrawn slowly to avoid hemolysis.
• Cannula/fistula/heparin lock - hospitals have special policies regarding these
devices. In general, blood should not be drawn from an arm with a fistula or
cannula without consulting the attending physician.
• Edematous extremities - tissue fluid accumulation alters test results.

PROCEDURE FOR VEIN SELECTION:

 Palpate and trace the path of veins with the index finger. Arteries pulsate, are most
elastic, and have a thick wall. Thrombosis veins lack resilience, feel cord-like, and roll
easily.
 If superficial veins are not readily apparent, you can force blood into the vein by
massaging the arm from wrist to elbow, tap the site with index and second finger, apply
a warm, damp washcloth to the site for 5 minutes, or lower the extremity over the
bedside to allow the veins to fill.

PERFORMANCE OF A VENIPUNCTURE:

➢ Approach the patient in a friendly, calm manner. Provide for their comfort as much as
possible, and gain the patient's cooperation.
➢ Identify the patient correctly.
➢ Properly fill out appropriate requisition forms, indicating the test(s) ordered.
➢ Verify the patient's condition. Fasting, dietary restrictions, medications, timing, and
medical treatment are all of concern and should be noted on the lab requisition.
➢ Check for any allergies to antiseptics, adhesives, or latex by observing for armbands
and/or by asking the patient.
➢ Position the patient. The patient should either sit in a chair, lie down or sit up in bed.
Hyperextend the patient's arm.
➢ Apply the tourniquet 3-4 inches above the selected puncture site. Do not place too tightly
or leave on more than 2 minutes (and no more than a minute to avoid increasing risk for
hem concentration). Wait 2 minutes before reapplying the tourniquet.
➢ The patient should make a fist without pumping the hand.
➢ Select the venipuncture site.
➢ Prepare the patient's arm using an alcohol prep. Cleanse in a circular fashion, beginning
at the site and working outward. Allow to air dry.
➢ Grasp the patient's arm firmly using your thumb to draw the skin taut and anchor the
vein. The needle should form a 15 to 30 degree angle with the surface of the arm. Swiftly
insert the needle through the skin and into the lumen of the vein. Avoid trauma and
excessive probing.
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➢ When the last tube to be drawn is filling, remove the tourniquet.

➢ Remove the needle from the patient's arm using a swift backward motion.
➢ Press down on the gauze once the needle is out of the arm, applying adequate pressure
to avoid formation of a hematoma.
➢ Dispose of contaminated materials/supplies in designated containers.
➢ Mix and label all appropriate tubes at the patient bedside.
➢ Deliver specimens promptly to the laboratory.

SAFETY AND INFECTION CONTROL

Because of contacts with sick patients and their specimens, it is important to follow
safety and infection control procedures.

PROTECT YOURSELF

o Practice universal precautions:

▪ Wear gloves and a lab coat or gown when handling blood/body fluids.
▪ Change gloves after each patient or when contaminated.
▪ Wash hands frequently.
▪ Dispose of items in appropriate containers.
▪ Dispose of needles immediately upon removal from the patient's vein. Do
not bend, break, recap needles to avoid accidental needle puncture or
splashing of contents.
▪ Clean up any blood spills with a disinfectant such as freshly made 10%
bleach.

If you stick yourself with a contaminated needle:

❖ Remove your gloves and dispose of them properly.

❖ Squeeze puncture site to promote bleeding.
❖ Wash the area well with soap and water.
❖ Record the patient's name and ID number.
❖ Follow institution's guidelines regarding treatment and follow-up.
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❖ NOTE: The use of prophylactic zidovudine following blood exposure to HIV has
shown effectiveness (about 79%) in preventing serum conversion

PROTECT THE PATIENT

• Place blood collection equipment away from patients, especially children and
psychiatric patients.
• Practice hygiene for the patient's protection. When wearing gloves, change them
between each patient and wash your hands frequently. Always wear a clean lab
coat or gown.

TROUBLESHOOTING GUIDELINES:

IF AN INCOMPLETE COLLECTION OR NO BLOOD IS OBTAINED:

➢ Change the position of the needle. Move it forward (it may not be in the lumen)

➢ Move it backward (it may have penetrated too far).

➢ Adjust the angle (the bevel may be against the vein wall).
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➢ Loosen the tourniquet. It may be obstructing blood flow.

➢ Try another tube. Use a smaller tube with fewer vacuums. There may be no vacuum in
the tube being used.
➢ Re-anchor the vein. Veins sometimes roll away from the point of the needle and
puncture site.
➢ Have the patient make a fist and flex the arm, which helps engorge muscles to fill veins.
➢ Pre-warm the region of the vein to reduce vasoconstriction and increase blood flow.
➢ Have the patient drink fluids if dehydrated.

BLOOD COLLECTION ON BABIES:

➢ The recommended location for blood collection on a newborn baby or infant is the heel.
The diagram below indicates in green the proper area to use for heel punctures for blood
collection:

➢ Pre warming the infant's heel (42 C for 3 to 5 minutes) is important to obtain capillary
blood gas samples and warming also greatly increases the flow of blood for collection of
other specimens. However, do not use too high a temperature warmer, because baby's
skin is thin and susceptible to thermal injury.
➢ Clean the site to be punctured with an alcohol swab. Dry the cleaned area with a dry
cotton sponge. Hold the baby's foot firmly to avoid sudden movement.
➢ Using a sterile blood lancet, puncture the side of the heel in the appropriate regions
shown above in green. Do not use the central portion of the heel because you might
injure the underlying bone, which is close to the skin surface. Do not use a previous
puncture site. Make the cut across the heel print lines so that a drop of blood can well up
and not run down along the lines.
➢ Wipe away the first drop of blood with a piece of clean, dry cotton. Since newborns do
not often bleed immediately, use gentle pressure to produce a rounded drop of blood. Do
not use excessive pressure or heavy massaging because the blood may become diluted
with tissue fluid.
➢ Fill the capillary tube(s) or micro collection device(s) as needed.
➢ When finished, elevate the heel, place a piece of clean, dry cotton on the puncture site,
and hold it in place until the bleeding has stopped.
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➢ Be sure to dispose of the lancet in the appropriate sharps container. Dispose of

contaminated materials in appropriate waste receptacles. Remove your gloves and wash
your hands.

COLLECTION TUBES FOR PHLEBOTOMY

• Collection tubes can vary in size for volume of blood drawn, appropriate to the tests
ordered with sample size required, and vary in the kind of additive for anticoagulation,
separation of plasma, or preservation of analyte. Larger tube sizes typically provide for
collection of samples from 6 to 10 ml.

Smaller collection tubes for sample sizes of 2 mL or less may be appropriate in situations
where a smaller amount blood should be drawn, as in pediatric patients, or to minimize
hemolysis during collection, or to avoid insufficient sample volume in the collection tube.

Red Top

ADDITIVE None

MODE OF Blood clots, and the serum is

ACTION separated by centrifugation

Chemistries, Immunology and

USES
Serology, Blood Bank (Crossmatch)
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Gold Top

ADDITIVE None

Serum separator tube (SST) contains a

MODE OF
gel at the bottom to separate blood
ACTION
from serum on centrifugation

USES Chemistries, Immunology and Serology

Light
Green
Top

Plasma Separating Tube (PST) with

ADDITIVE
Lithium heparin

Anticoagulants with lithium heparin;

MODE OF
Plasma is separated with PST gel at the
ACTION
bottom of the tube

USES Chemistries

Purple Top

ADDITIVE EDTA

MODE OF
Forms calcium salts to remove calcium
ACTION

Hematology and Blood Bank

USES (Crossmatch);requires fulldraw invert 8
times to prevent clotting and clumping.
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Light
Blue Top

ADDITIVE Sodium citrate

MODE
OF Forms calcium salts to remove calcium
ACTION

Coagulation tests (protime and

USES
prothrombin time), full draw required

Light
Gray Top

ADDITIVE Sodium fluoride and potassium oxalate

MODE
Anti glycolytic agent preserves glucose
OF
up to 5 days
ACTION

Glucoses, requires full draw (may cause

USES
hemolysis if short draw)

Green Top

ADDITIVE Sodium heparin or lithium heparin

MODE OF Inactivates thrombin and

ACTION thromboplastin
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For lithium level, use sodium heparin

USES For ammonia level, use sodium or
lithium heparin

Yellow
Top

ADDITIVE ACD (acid-citrate-dextrose)

MODE OF
Complement inactivation
ACTION

HLA tissue typing, paternity testing,

USES
DNA studies

Yellow -
Black Top

ADDITIVE Broth mixture

MODE OF
Preserves viability of microorganisms
ACTION

Microbiology - aerobes, anaerobes,

USES
fungi
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Black Top

ADDITIVE Sodium citrate (buffered)

MODE OF
Forms calcium salts to remove calcium
ACTION

Westergren Sedimentation Rate;

USES
requires full draw

Orange Top

ADDITIVE Thrombin

MODE OF
Quickly clots blood
ACTION

USES STAT serum chemistries

Light
Brown
Top

ADDITIVE Sodium heparin

MODE Inactivates thrombin and

OF thromboplastin; contains virtually no
ACTION lead

USES Serum lead determination

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Pink Top

ADDITIVE Potassium EDTA

MODE OF
Forms calcium salts
ACTION

USES Immunohematology

White Top

ADDITIVE Potassium EDTA

MODE OF
Forms calcium salts
ACTION

USES Molecular/PCR and bDNA testing