SPECIMEN

COLLECTION
 SPECIMEN CONSIDERATIONS

 SPECIMEN COLLECTION, HANDLING,

AND PROCESSING REMAINS ONE OF
THE PRIMAY AREAS OF
PREANALYTICAL ERROR.
 CAREFUL ATTENTION TO EACH
PHASE IS NECESSARY TO ENSURE
PROPER SUBSEQUENT TESTING AND
REPORTING OF MEANINGFUL
RESULTS
 SPECIMEN CONSIDERATIONS

 SPECIMEN COLLECTION, HANDLING,

AND PROCESSING REMAINS ONE OF
THE PRIMAY AREAS OF
PREANALYTICAL ERROR.
 CAREFUL ATTENTION TO EACH
PHASE IS NECESSARY TO ENSURE
PROPER SUBSEQUENT TESTING AND
REPORTING OF MEANINGFUL
RESULTS
 TYPES OF SAMPLE

 WHOLE BLOOD
 PLASMA: centrifuge whole blood at
RCF of 1000g to 2000g for 10
minutes
 ARTERIAL BLOOD: for blood gases
 SERUM:
 Other body fluids: CSF, paracentesis
fluids, amniotic fluids
 OBJECTIVES

 Describe and perform the venipuncture process including:

 Proper patient identification procedures.
 Proper equipment selection and use.
 Proper labeling procedures and completion of laboratory
requisitions.
 Order of draw for multiple tube phlebotomy.
 Preferred venous access sites, and factors to consider in site
selection, and ability to differentiate between the feel of a
vein, tendon and artery.
 Patient care following completion of venipuncture.
 Safety and infection control procedures.
 Quality assurance issues.
 OBJECTIVES

 Identify the additive, additive function, volume, and specimen

considerations to be followed for each of the various color
coded tubes.
 List six areas to be avoided when performing venipuncture and
the reasons for the restrictions.
 Summarize the problems that may be encountered in accessing
a vein, including the procedure to follow when a specimen is
not obtained.
 List several effects of exercise, posture, and tourniquet
application upon laboratory values.
 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. Several essential steps
are required for every successful collection procedure:
 Identify the patient.
 Assess the patient's physical disposition (i.e. diet, exercise, stress, basal
state).
 Check the requisition form for requested tests, patient information, and
any special requirements.
 Select a suitable site for venipuncture.
 Prepare the equipment, the patient and the puncture site.
 Perform the venipuncture.
 Collect the sample in the appropriate container.
 Recognize complications associated with the phlebotomy procedure.
 Assess the need for sample recollection and/or rejection.
 Label the collection tubes at the bedside or drawing area.
 Promptly send the specimens with the requisition to the laboratory.
 ORDER FORM/REQUISITION

 A requisition form must accompany each sample submitted to

the laboratory. This requisition form must contain the proper
information in order to process the specimen. The essential
elements of the requisition 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.
 Initials of phlebotomist.
 Indicating the test(s) requested.
 An example of a simple requisition form with the essential
elements is shown below:
 LABELING THE SAMPLE
 A properly labelled sample is essential so
that the results of the test match the
patient. The key elements in labelling are:
 Patient's surname, first and middle.
 Patient's ID number.
 NOTE: Both of the above MUST match the same on
the requisition form.
 Date, time and initials of the phlebotomist must
be on the label of EACH tube.
 An example of a simple requisition form with
the essential elements is shown below:
 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 (see illustrations at end).
 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 Wipes - 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.
 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 - may be 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 is:
 First - blood culture tube (yellow-black stopper)
 Second - non-additive tube (red stopper or SST)
 Third - coagulation tube (light blue stopper). If just a
routine coagulation assay is the only test ordered, then
a single light blue stopper 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 stopper tube.
 ORDER OF DRAW
 Last draw - additive tubes in this order:
 Last draw - additive tubes in this order:
 SST (red-gray, or gold, stopper). Contains a gel separator and
clot activator.
 Sodium heparin (dark green stopper)
 PST (light green stopper). Contains lithium heparin
anticoagulant and a gel separator.
 EDTA (lavender stopper)
 ACDA or ACDB (pale yellow stopper). Contains acid citrate
dextrose.
 Oxalate/fluoride (light gray stopper)
 NOTE:Tubes with additives must be thoroughly
mixed. Erroneous test results may be obtained when
the blood is not thoroughly mixed with the additive.
 NOTE:For plastic tubes, the order of draw for tubes 2
and 3 is reversed.
 PROCEDURAL ISSUES

 PATIENT RELATIONS AND IDENTIFICATION:

 The phlebotomist's role requires a professional,
courteous, and understanding manner in all contacts
with the patient. Greet the patient and identify
yourself and indicate the procedure that will take
place. Effective communication - both verbal and
nonverbal - is essential.
 Proper patient identification MANDATORY. If an
inpatient is able to respond, ask for a full name and
always check the armband for confirmation. DO NOT
DRAW BLOOD IF THE ARMBAND IS MISSING. An
outpatient must provide identification other than the
verbal statement of a name. Using the requisition for
reference, ask a patient to provide additional
information such as a surname or birthdate.
 PROCEDURAL ISSUES

 If possible, speak with the patient during the

process. The patient who is at ease will be less
focused on the procedure. Always thank the patient
and excuse yourself courteously when finished.
 PATIENT'S BILL OF RIGHTS:
 The Patient's Bill of Rights has been adopted by
many hospitals as declared by the Joint Commission
on Accreditation of Healthcare Organizations
(JCAHO). The basic patient rights endorsed by the
JCAHO follow in condensed form are given below.
 The patient has the right to:
 Impartial access to treatment or accommodations
that are available or medically indicated,
regardless of race, creed, sex, national origin, or
sources of payment for care.
 Considerate, respectful care.
 Confidentiality of all communications and other
records pertaining to the patient's care.
 Expect that any discussion or consultation
involving the patient's case will be conducted
discreetly and that individuals not directly
involved in the case will not be present without
patient permission.
 Expect reasonable safety congruent with the
hospital practices and environment.
 Know the identity and professional status of
individuals providing service and to know
which physician or other practitioner is
primarily responsible for his or her care.
 Obtain from the practitioner complete and
current information about diagnosis,
treatment, and any known prognosis, in
terms the patient can reasonably be
expected to understand.
 Reasonable informed participation in decisions
involving the patient's health care. The patient shall
be informed if the hospital proposes to engage in or
perform human experimentation or other
research/educational profits affecting his or her care
or treatment. The patient has the right to refuse
participation in such activity.
 Consult a specialist at the patient's own request and
expense.
 Refuse treatment to the extent permitted by law.
 Regardless of the source of payment, request and
receive an itemized and detailed explanation of the
total bill for services rendered in the hospital.
 Be informed of the hospital rules and regulations
regarding patient conduct.
 VENIPUNCTURE SITE SELECTION:
 Although the larger and fuller median cubital and
cephalic veins of the arm are used most frequently,
wrist and hand veins are also acceptable for
venipuncture.
 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:
 Turn off the IV for at least 2 minutes
before venipuncture.
 Apply the tourniquet below the IV site.
Select a vein other than the one with
the IV.
 Perform the venipuncture. Draw 5 ml of
blood and discard before drawing the
specimen tubes for testing.
 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. Thrombosed
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.
 PERFORMANCE OF A
VENIPUNCTURE
 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.
 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.
 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.
 PERFORMANCE OF A FINGERSTICK:

 Follow the procedure as outlined above for greeting and

identifying the patient. As always, 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.
 Position the patient. The patient should either sit in a chair, lie
down or sit up in bed. Hyperextend the patient's arm.
 The best locations for fingersticks are the 3rd and 4th fingers of
the non-dominant hand. Do not use the tip of the finger or the
center of the finger. Avoid the side of the finger where there is
less soft tissue, where vessels and nerves are located, and where
the bone is closer to the surface. The 2nd (index) finger tends to
have thicker, callused skin. The fifth finger tends to have less soft
tissue overlying the bone. Avoid puncturing a finger that is cold or
cyanotic, swollen, scarred, or covered with a rash.
 PERFORMANCE OF A FINGERSTICK:
 Using a sterile lancet, make a skin puncture just off the
center of the finger pad. The puncture should be made
perpendicular to the ridges of the fingerprint so that the drop
of blood does not run down the ridges.
 Wipe away the first drop of blood, which tends to contain
excess tissue fluid.
 Collect drops of blood into the collection device by gently
massaging the finger. Avoid excessive pressure that may
squeeze tissue fluid into the drop of blood.
 Cap, rotate and invert the collection device to mix the blood
collected.
 Have the patient hold a small gauze pad over the puncture
site for a couple of minutes to stop the bleeding.
 Dispose of contaminated materials/supplies in designated
containers.
 Label all appropriate tubes at the patient bedside.
 Deliver specimens promptly to the laboratory.
ADDITIONAL CONSIDERATIONS:
 To prevent a hematoma:
 Puncture only the uppermost wall of the vein
 Remove the tourniquet before removing the needle
 Use the major superficial veins
 Make sure the needle fully penetrates the upper most wall of the
vein. (Partial penetration may allow blood to leak into the soft
tissue surrounding the vein by way of the needle bevel)
 Apply pressure to the venipuncture site
 To prevent hemolysis (which can interfere with many tests):
 Mix tubes with anticoagulant additives gently 5-10 times
 Avoid drawing blood from a hematoma
 Avoid drawing the plunger back too forcefully, if using a needle and
syringe, and avoid frothing of the sample
 Make sure the venipuncture site is dry
 Avoid a probing, traumatic venipuncture
 Indwelling Lines or Catheters:
 Potential source of test error
 Most lines are flushed with a solution of heparin to reduce the
risk of thrombosis
 Discard a sample at least three times the volume of the line
before a specimen is obtained for analysis
 Hemoconcentration: An increased concentration of larger
molecules and formed elements in the blood may be due to
several factors:
 Prolonged tourniquet application (no more than 2 minutes)
 Massaging, squeezing, or probing a site
 Long-term IV therapy
 Sclerosed or occluded veins
 Prolonged Tourniquet Application:
 Primary effect is hemoconcentration of non-filterable
elements (i.e. proteins). The hydrostatic pressure causes
some water and filterable elements to leave the extracellular
space.
 Significant increases can be found in total protein, aspartate
aminotransferase (AST), total lipids, cholesterol, iron
 Affects packed cell volume and other cellular elements
  Patient Preparation Factors:
 Therapeutic Drug Monitoring: different
pharmacologic agents have patterns of
administration, body distribution, metabolism, and
elimination that affect the drug concentration as
measured in the blood. Many drugs will have
"peak" and "trough" levels that vary according to
dosage levels and intervals. Check for timing
instructions for drawing the appropriate samples.
 Effects of Exercise: Muscular activity has both
transient and longer lasting effects. The creatine
kinase (CK), aspartate aminotransferase (AST),
lactate dehydrogenase (LDH), and platelet count
may increase.
 Stress: May cause transient elevation in white
blood cells (WBC's) and elevated adrenal hormone
values (cortisol and catecholamines). Anxiety that
results in hyperventilation may cause acid-base
imbalances, and increased lactate.

 Diurnal Rhythms: Diurnal rhythms are body fluid
and analyte fluctuations during the day. For
example, serum cortisol levels are highest in
early morning but are decreased in the
afternoon. Serum iron levels tend to drop during
the day. You must check the timing of these
variations for the desired collection point.
 Posture: Postural changes (supine to sitting etc.)
are known to vary lab results of some analytes.
Certain larger molecules are not filterable into
the tissue, therefore they are more concentrated
in the blood. Enzymes, proteins, lipids, iron, and
calcium are significantly increased with changes
in position.
 Other Factors: Age, gender, and pregnancy have
an influence on laboratory testing. Normal
reference ranges are often noted according to
age.
 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
 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, or resheath 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.
 NOTE: The use of prophylactic zidovudine
following blood exposure to HIV has shown
effectiveness (about 79%) in preventing
seroconversion
 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.
 IF BLOOD STOPS FLOWING INTO THE TUBE:
 The vein may have collapsed; resecure the
tourniquet to increase venous filling. If this is not
successful, remove the needle, take care of the
puncture site, and redraw.
 The needle may have pulled out of the vein when
switching tubes. Hold equipment firmly and place
fingers against patient's arm, using the flange for
leverage when withdrawing and inserting tubes.
 PROBLEMS OTHER THAN AN INCOMPLETE
COLLECTION:
 A hematoma forms under the skin adjacent to the
puncture site - release the tourniquet immediately
and withdraw the needle. Apply firm pressure.
 The blood is bright red (arterial) rather than
venous. Apply firm pressure for more than 5
minutes.
 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)
 or move it backward (it may have penetrated too
far).
 Adjust the angle (the bevel may be against the vein
wall).
 Loosen the tourniquet. It may be obstructing blood
flow.
 Try another tube. There may be no vacuum in the
one being used.
 Re-anchor the vein. Veins sometimes roll away from
the point of the needle and puncture site.
 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:
 Prewarming the infant's heel (42 C for 3 to 5 minutes) is
important to obtain capillary blood 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 sponge. Dry
the cleaned area with a dry cotton sponge. Hold the baby's
foot firmly to avoid sudden movement.
 BLOOD COLLECTION ON BABIES:
 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 heelprint 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.
 BLOOD COLLECTION ON BABIES:
 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.
 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.

Red Top ADDITIVE None
MODE OF ACTION Blood
clots, and the serum is
separated by centrifugation
USES Chemistries,
Immunology and Serology,
Blood Bank (Crossmatch)
 Gold Top ADDITIVE None MODE OF
ACTION Serum separator tube (SST) contains
a gel at the bottom to separate blood from
serum on centrifugation USES Chemistries,
Immunology and Serology
 Light Green Top ADDITIVE Plasma
Separating Tube (PST) with Lithium heparin
MODE OF ACTION Anticoagulates with lithium
heparin; Plasma is separated with PST gel at
the bottom of the tube USES Chemistries
 Red-Gray Top ADDITIVE Serum Separating
Tube (SST) with clot activator MODE OF
ACTION Forms clot quickly and separates the
serum with SST gel at the bottom of the tube
USES Chemistries
 Purple Top ADDITIVE EDTA liquid MODE
OF ACTION Forms calcium salts to remove
calcium USES Hematology (CBC) and Blood
Bank (Crossmatch); requires full draw -
invert 8 times to prevent clotting and platelet
clumping
 Light Blue Top ADDITIVE Sodium citrate
MODE OF ACTION Forms calcium salts to
remove calcium USES Coagulation tests
(protime and prothrombin time), full draw
required
 Dark Green Top ADDITIVE Sodium heparin
or lithium heparin MODE OF ACTION
Inactivates thrombin and thromboplastin
USES For lithium level, use sodium heparin
For ammonia level, use sodium or lithium
heparin
 Dark Blue Top ADDITIVE Sodium EDTA
MODE OF ACTION Tube is designed to
contain no contaminating metals USES Trace
element testing (zinc, copper, lead, mercury)
and toxicology
 Light Gray Top ADDITIVE Sodium fluoride
and potassium oxalate MODE OF ACTION
Antiglycolytic agent preserves glucose up to 5
days USES For lithium level, use sodium
heparin
Glucoses, requires full draw (may cause
hemolysis if short draw)
 Yellow Top ADDITIVE ACD (acid-citrate-
dextrose) MODE OF ACTION Complement
inactivation USES HLA tissue typing, paternity
testing, DNA studies
 Yellow - Black Top ADDITIVE Broth
mixture MODE OF ACTION Preserves viability
of microorganisms USES Microbiology -
aerobes, anaerobes, fungi
 Black Top ADDITIVE Sodium citrate
(buffered) MODE OF ACTION Forms calcium
salts to remove calcium USES Westergren
Sedimentation Rate; requires full draw
 Orange Top ADDITIVE Thrombin MODE OF
ACTION Quickly clots blood USES STAT serum
chemistries
 Brown Top ADDITIVE Sodium heparin
MODE OF ACTION Inactivates thrombin and
thromboplastin USES Serum lead
determination
 SAMPLE PROCESSING
 GENERALLY, SAMPLES SHOULD BE ANALYZED
WITHIN 4 HOURS AND TO MINIMIZE THE
EFFECT OF EVAPORATION, THEY SHOULD BE
PROPERLY CAPPED, AWAY FROM AREAS OF
RAPID AIRFLOW, LIGHT AND HEAT.
 REFRIGERATION AT 4C FOR 8h, MOST
ANALYTES ARE QUITE STABLE EXCEPT
ALP(INCREASE) AND LDH(DECREASE)
 FROZEN AT -20C
 SAMPLE VARIABLES
 Physiologic considerations
 Proper patient preparation
 Problems in collection
 Transportation
 Processing
 Storage
 CHAIN OF CUSTODY
 TESTS ARE LINKED TO CRIME OR ACCIDENT
 DOCUMENT SPECIMEN IDENTIFICATION AND
PHASE OF PROCESS
 TAMPER-PROOF SEAL
 WITNESS
 CO-SIGN