Clinical Chemistry 1: Phlebotomy

VENIPUNCTURE PROCEDURE 3. Non-additive tube (red top)

1. Identify the patient. 4. Additive tubes in this order:
2. Assess the patient's physical disposition (i.e. diet, a. SST (red-gray or gold top)
exercise, stress, basal state). b. Sodium heparin (dark green top)
3. Check the requisition form for requested tests, patient c. PST (light green top)
information, and any special requirements. d. EDTA (lavender top)
4. Select a suitable site for venipuncture. e. ACDA or ACDB (pale yellow top)
5. Prepare the equipment, the patient and the puncture f. Oxalate/fluoride (light gray top
site. PROCEDURAL ISSUES
6. Perform the venipuncture. ✓ Patient relations and identification
7. Collect the sample in the appropriate container. ✓ The phlebotomist's role requires a professional,
8. Recognize complications associated with the courteous, and understanding manner in all contacts
phlebotomy procedure. with the patient.
9. Assess the need for sample recollection and/or ✓ Proper patient identification mandatory.
rejection. ✓ An outpatient must provide identification other than the
10. Label the collection tubes at the bedside or drawing verbal statement of a name.
area. RIGHTS
11. Promptly send the specimens with the requisition to the 1. Impartial access to treatment or accommodations that
laboratory. are available or medically indicated, regardless of race,
ORDER FORM/REQUISITION creed, sex, national origin, or sources of payment for
✓ Patient's surname, first name, and middle initial. care.
✓ Patient's ID number. 2. Considerate, respectful care.
✓ Patient's date of birth and sex. 3. Confidentiality of all communications and other records
✓ Requesting physician's complete name. pertaining to the patient's care.
✓ Source of specimen- information must be given when 4. Expect reasonable safety congruent with the hospital
requesting microbiology, cytology, fluid analysis, or practices and environment.
other testing where analysis and reporting is site 5. Know the identity and professional status of individuals
specific. providing service.
✓ Date and time of collection. 6. Reasonable informed participation in decisions involving
✓ Initials of phlebotomist. the patient's health care.
✓ Indicating the test(s) requested. 7. Consult a specialist at the patient's own request and
LABELING THE SAMPLE expense.
✓ Patient's surname, first and middle. 8. Refuse treatment to the extent permitted by law.
✓ Patient's ID number. 9. Regardless of the source of payment, request and
NOTE: Both of the above MUST match the same on the receive an itemized and detailed explanation of the total
requisition form. bill for services rendered in the hospital.
✓ Date, time and initials of the phlebotomist must be on 10. Be informed of the hospital rules and regulations
the label of EACH tube. regarding patient conduct
✓ Automated systems may include labels with bar codes. VENIPUNCTURE SITE SELECTION
1. Median cubital
2. Cephalic veins
3. Basilic vein
4. Dorsal hand veins
NEEDED FOR ROUTINE VENIPUNCTURE: 5. Foot veins
1. Evacuated Collection Tubes Certain areas are to be avoided when choosing a site:
2. Needles ✓ Extensive scars from burns and surgery
3. Holder/Adapter ✓ The upper extremity on the side of a previous
4. Tourniquet mastectomy
5. Alcohol Wipes ✓ Hematoma
6. Povidone-iodine wipes/swabs ✓ Intravenous therapy (IV) / blood transfusions
7. Gauze sponges ✓ Cannula/fistula/heparin lock
8. Adhesive bandages/tape ✓ Edematous extremities
9. Needle disposal unit PROCEDURE FOR VEIN SELECTION:
10. Gloves 1. Palpate and trace the path of veins with the index finger.
11. Syringes Arteries pulsate, are most elastic, and have a thick wall.
ORDER OF DRAW: Thrombosed veins lack resilience, feel cord-like, and roll
1. Blood culture bottle or tube (yellow or yellow-black top) easily.
2. Coagulation tube (light blue top)

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 Clinical Chemistry 1: Phlebotomy

2. If superficial veins are not readily apparent, you can fingers against patient's arm, using the flange for
force blood into the vein by massaging the arm from leverage when withdrawing and inserting tubes.
wrist to elbow, tap the site with index and second finger, Problems other than an incomplete collection:
apply a warm, damp washcloth to the site for 5 minutes, 1. A hematoma forms under the skin adjacent to the
or lower the extremity over the bedside to allow the puncture site - release the tourniquet immediately and
veins to fill. withdraw the needle. Apply firm pressure.
Additional considerations:
✓ To prevent a hematoma
✓ To prevent hemolysis (which can interfere with many 2. The blood is bright red (arterial) rather than venous.
tests) Apply firm pressure for more than 5 minutes.
✓ Indwelling Lines or Catheters
✓ Hemoconcentration
✓ Prolonged Tourniquet Application
Patient Preparation Factors
✓ Therapeutic Drug Monitoring Color Additive Mode Of Action Uses
✓ Effects of Exercise Red None Blood clots, and Chemistries,
✓ Stress the serum is Immunology
✓ Diurnal Rhythms separated by and Serology,
✓ Posture centrifugation Blood Bank
✓ Other Factors (Crossmatch)
SAFETY AND INFECTION CONTROL Gold None Serum separator Chemistries,
Protect yourself tube (SST) Immunology
• Practice universal precautions contains a gel at and Serology
• Dispose of needles immediately the bottom to
• Clean up any blood spills with a disinfectant such as separate blood
freshly made 10% bleach. from serum on
• If you stick yourself with a contaminated needle: centrifugation
Protect the patient Light Plasma Anticoagulates Chemistries
• Place blood collection equipment away from patients, Green Separating with lithium
especially children and psychiatric patients. Top Tube (PST) heparin; Plasma
• Practice hygiene for the patient's protection. with is separated with
TROUBLESHOOTING GUIDELINES Lithium PST gel at the
1. If an incomplete collection or no blood is obtained heparin bottom of the
✓ Change the position of the needle. tube
✓ Move it forward (it may not be in the lumen) or Purple EDTA Forms calcium Hematology
move it backward (it may have penetrated too Top salts to remove (CBC) and Blood
far). calcium Bank
✓ Adjust the angle (the bevel may be against the (Crossmatch);
vein wall). requires full
✓ Loosen the tourniquet. draw - invert 8
✓ Try another tube. There may be no vacuum in times to prevent
the one being used. clotting and
✓ Re-anchor the vein. Veins sometimes roll away platelet
from the point of the needle and puncture site. clumping
Light Sodium Forms calcium Coagulation
Blue citrate salts to remove tests (protime
Top calcium and
prothrombin
time), full draw
required
If blood stops flowing into the tube: Green Sodium Inactivates For lithium
✓ The vein may have collapsed; resecure the tourniquet to Top heparin or thrombin and level, use
increase venous filling. If this is not successful, remove lithium thromboplastin sodium heparin
the needle, take care of the puncture site, and redraw. heparin For ammonia
✓ The needle may have pulled out of the vein when level, use
switching tubes. Hold equipment firmly and place sodium or
lithium heparin

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 Clinical Chemistry 1: Phlebotomy

Dark EDTA Tube is designed Trace element ✓ Fill the capillary tube(s) or micro collection device(s) as
Blue to contain no testing (zinc, needed.
Top contaminating copper, lead, ✓ When finished, elevate the heel, place a piece of clean,
metals mercury) and dry cotton on the puncture site, and hold it in place until
toxicology the bleeding has stopped.
Light Sodium Antiglycolytic Glucoses, ✓ Be sure to dispose of the lancet in the appropriate
Gray fluoride agent preserves requires full sharps container. Dispose of contaminated materials in
Top and glucose up to 5 draw (may appropriate waste receptacles. Remove your gloves and
potassium days cause hemolysis wash your hands.
oxalate if short draw)
Yellow ACD (acid- Complement HLA tissue
Top citrate- inactivation typing, paternity
dextrose) testing, DNA
studies
Yellow Broth Preserves Microbiology -
- Black mixture viability of aerobes,
Top microorganisms anaerobes,
fungi
Black Sodium Forms calcium Westergren
Top citrate salts to remove Sedimentation
(buffered) calcium Rate; requires
full draw
Orange Thrombin Quickly clots STAT serum
Top blood chemistries
Light Sodium Inactivates Serum lead
Brown heparin thrombin and determination
Top thromboplastin;
contains virtually
no lead
Pink Potassium Forms calcium Molecular/viral
Top EDTA salts load testing

BLOOD COLLECTION ON BABIES

✓ The recommended location for blood collection on a
newborn baby or infant is the heel.
✓ Prewarming 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 sponge.
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 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.

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 Clinical Chemistry 1: Phlebotomy

PHLEBOTOMY AND SPECIMEN CONSIDERATION 4. Legal issues

✓ The collection, handling, and processing of specimens ✓ The most common civil actions in healthcare
represent a critical step in specimen analysis. are based on “tort” an intentional or
✓ Physicians rely on results obtained from quality unintentional wrong or act that is committed
laboratory specimens to confirm health or diagnose and without just cause.
treat patients. ✓ Examples of tort actions that could involve
✓ The most sophisticated laboratory equipment cannot phlebotomists are as follows:
deliver valid results if specimen integrity is a. Assault: a threat that causes one to be
compromised. in fear of immediate harm
PUBLIC RELATION AND CLIENT INTERACTION. b. Battery: deliberate harmful or
1. Professionalism offensive touching without consent or
a. Appearance legal justification
b. Communication Skills c. Invasion of privacy: violation of one’s
c. Bedside Manner right to be left alone
d. Attitude d. Breach of confidentiality: failure to
✓ Integrity & honesty keep private or privileged information
✓ Compassion confidential
✓ Motivation e. Negligence: doing something that a
✓ Dependability & Work ethic reasonable person would not do or
✓ Diplomacy not doing something that a reasonable
✓ Ethical Behavior person would do
2. Patient consent f. Malpractice: a type of negligence
Informed Implies voluntary permission for a medical committed by a professional
consent procedure, test, or medication will be given g. Standard of care: a duty to protect
Expressed someone from harm established by
May be given verbally or in writing
consent standards of the profession and
Does not require a verbal expression of expectations of society
Implied consent. Actions can imply consent (e.g., the 5. Infection control
consent patient holds out an arm after being told a ✓ Standard precautions must be taken with every
blood specimen is to be collected) patient to prevent the spread of infection. This
Laws specify exactly what type of includes wearing personal protective
HIV consent information must be given to inform the equipment (PPE) when drawing blood or
client properly handling specimens and using proper hand
Parent or guardian consent is required hygiene procedures. Additional transmission-
Consent for based precautions (i.e., airborne, droplet, or
(health care personnel who do not obtain it
minors contact) may be required for patients with
are liable for assault and battery)
An individual has a constitutional right to certain diseases.
Refusal of PATIENT IDENTIFICATION PROCEDURES
refuse a medical procedure such as
consent ✓ Verbally ask their full names
venipuncture
including middle names
Conscious II-
3. Confidentiality and the Health Insurance Portability ✓ Verify the name using the
patients /
and Accountability Act identification bracelet which
Hospitalized
- Maintaining patient confidentiality protects includes full names, hospital
patients
patients and practitioners. Health care workers are number, room number, bed
required to treat all patient information as private number, physician’s name, etc.
and confidential. Because it can now be easily ✓ Identified in the same manner as
transmitted electronically, the Health Insurance conscious patients
Sleeping patients
Portability and Accountability Act (HIPAA) was ✓ Must be awakened before blood
passed to more closely secure patient information. collection
This federal law requires that patients be given Unconscious / ✓ They are identified by asking the
information on their rights concerning the release Mentally attending nurse or relative; ID
of protected health information (PHI) and on how it incompetent Bracelet
will be used. All health care workers must sign a patients
special form stating they understand HIPAA rules ✓ A nurse or relative may identify the
Infants and
and will keep all patient information confidential. patient, or by means of an
Children
Penalties for HIPAA violations include disciplinary identification bracelet.
action, fines, and possible jail time.

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 Clinical Chemistry 1: Phlebotomy

Outpatient / 2) Have the patient open his/her fist, and observe

Ambulatory
patient
3-way ID System
✓ To avoid misidentification, a phlebotomist nay require
what is referred to as 3-way ID, in which the patient is
identified by:
a. The patient’s verbal ID statement
b. A check on the ID band
c. A visual comparison of the labeled specimen
with the patient’s ID band before leaving the
bedside
PHLEBOTOMY
- process of collecting blood
- meaning “to cut a vein”
- performed by Phlebotomist
Types of Phlebotomy: Arterial Puncture, Venipuncture, Capillary
Puncture
ARTERIAL PUNCTURE
- process by which blood is obtained from a patient’s
artery.
- arterial blood is the oxygenated blood with a bright red
color
Uses:
a. Blood Gas Analysis (partial pressure of oxygen and
carbon dioxide)
b. pH measurement
Sites:
a. Radial artery
b. Brachial artery
c. Femoral artery (in order of preference)
d. Scalp artery
e. Umbilical artery
NOTES:
✓ Radial artery puncture can be painful and associated
with symptoms such as aching, throbbing, tenderness,
sharp sensation, and cramping.
✓ The femoral artery is relatively large and easy to
puncture, but extra care must be given to older
individuals because the femoral artery can bleed more
than the radial or brachial.
✓ Arterial bleeding is the hardest to control and usually
requires special attention.
✓ Major complications: thrombosis, hemorrhage, and
possible infection.
✓ Unacceptable sites: irritated, edematous, near a wound,
or in an area of an arteriovenous shunt or fistula
✓ Before blood is collected from the radial artery,
modified Allen test should be done to determine
whether the ulnar artery can provide collateral
circulation to the hand after the radial artery puncture.
Modified Allen Test
1) Have the patient make a fist and occlude both
the ulnar (opposite the thumb side) and the
radial arteries (closest to the thumb) by
compressing with two fingers over each artery.
if the patient’s palm has become bleached of
blood.
3) Release the pressure on the ulnar artery
(farthest from the thumb) only and note if
blood return is present. The palm should
become perfused with blood. Adequate
perfusion is a positive test indicating that
arterial blood may be drawn from the radial
artery. Blood should not be taken if the test is
negative. Serious consequences may occur if
this procedure is not followed, which may
result in loss of the hand or its function.
Materials:
1. Glass needle
2. Cotton (dry and wet)
3. Alcohol or Iodine
4. Heparin –preferred anticoagulant
5. Warm towel (45’C) –arterializations
6. Iced water bath or other coolant (1-5’C –
preservative for transport) to minimize
leukocyte consumption of oxygen.
7. Each site of arterial puncture uses different
gauge of needle
a. Brachial artery: 18-20 G (45-60
degreeangle)
b. Radial artery: 23-25 G
c. Femoral artery (90 degree angle)
Procedure:
1) Prepare the arterial blood gas (ABG) syringe
according to established procedures. The
needle (18-20 gauge for brachial artery) should
pierce the skin at an angle of approx. 45-60
degrees (90 degrees for femoral artery) in a
slow and deliberate manner. Some dorsiflexion
of the wrist is necessary with the radial artery,
for which 23-25 gauge needle is used. The
pulsation of blood into the syringe confirm that
is will fill by arterial pressure alone. If the
plunger is pulled back and air is aspirated,
immediately withdraw the syringe.
2) After the blood specimen is collected, gently
rotate the syringe, mixing blood and heparin.
Place in ice water (or other coolant that will
maintain a temperature of 1-5’C) to minimize
leukocyte consumption of oxygen.
3) After the arterial puncture, compression with a
sterile gauze pad on the puncture site should
be applied for a minimum of 2 minutes and
preferable for 5 minutes. Apply an adhesive
bandage.
MORE NOTES:
✓ The artery to be punctured is identified by its
pulsations and cleansed with 70% aqueous
isopropanol solution followed by iodine.
✓ A non-anesthetized arterial puncture provides
an accurate measurement of resting Ph and

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 Clinical Chemistry 1: Phlebotomy

pCO2 in spite of the theoretical error possibly

from patient hyperventilation caused by the
pain of the arterial puncture.
✓ The use of butterfly infusion sets is no
recommended.
✓ Using19-gauge versus 25-gauge does not vary
the pCO2 or pO2 more than 1mmHg.
✓ The amount of anticoagulant should be 0.05mL
liquid heparin (1000IU/mL) for each milliliter of
blood.
✓ Using too much heparin is probably the most
common pre-analytic error in blood gas
measurement.
VENIPUNCTURE Veins on the wrist and dorsal aspects of hands
- In clinical laboratory, venipuncture is defined as all of - can be chosen provided that the antecubital vein is not
the steps involved in obtaining an appropriate and acceptable.
identified blood specimen from patient’s vein
- Collection of venous blood or deoxygenated blood (dark
red color)
NOTES:
✓ Before collecting a specimen, a phlebotomist should
dress in personal protective equipment (PPE), with
additional precautions and equipment or patients in
isolation as required by institutional policies.
✓ The phlebotomist should verify that the patient is
fasting, what medications are being taken or have been
discontinued as required, and so forth.
✓ The patient (1) should be comfortable, (2) should be
seated or supine (if sitting is not feasible), and (3) should
have been in this position or as long as possible before
the specimen is drawn.
✓ At no time should venipuncture be performed on a
standing patient. Either of the patient’s arms should be
extended in a straight line from the shoulder to the
wrist.
✓ An arm with an inserted intravenous line should be
avoided, as should an arm with extensive scarring or
with a hematoma at the intended collection site.
✓ If petechiae appear after venipuncture, it indicates that
minute amount of blood have escaped into skin
epithelium.
Sites:
Antecubital fossa region
Basilic vein - Should not be chosen unless no other vein
is more prominent due to its proximity to
the brachial artery
- It tends to roll more easily.
Cephalic - second choice if the median cubital vein is
vein unsuitable -
- it rolls and bruises less easily, but the blood
flows more easily Veins on the ankle
- it is also tougher to puncture - should be used only if arm veins have been determined
Median - is the best site for venipuncture because it to be unsuitable.
cubital is the largest and the best anchored vein
vein - has good blood flow and bruising is less
common

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 Clinical Chemistry 1: Phlebotomy

✓ If the surgery was done within 6months on both sides, a

vein on the back of the hand or at the ankle should be
used.
✓ The veins on the back of the hand or at the ankle are less
desirable and should be AVOIDED in diabetic patients
and other who have poor circulation.
✓ The torniquet must be applied 3-4 inches above the site.
✓ Torniquet application should not exceed 1 minute.
✓ If blood pressure cuff is used as torniquet, the pressure
must be 40mmHg.
Steps: ✓ Insert the needle (bevel up) into the skin at a 15–20-
1. Check the test request degree angle.
2. Approach, identify and prepare patient ✓ Most commonly used needle gauges: 19, 20, 21, and 22
3. Verify diet restrictions and latex sensitivity (the larger the gauge, the smaller the bore)
4. Sanitize hands ✓ Routine venipuncture uses a 23- or 21-gauge needle.
5. Position patient, apply torniquet, and ask patient to ✓ Most commonly used needle length: 1 to 1½ inches
make fist. (2.5to3.7cm)
6. Select vein, release torniquet and ask patient to open ✓ Traces of alcohol on the skin may cause hemolysis.
fist. ✓ For ethanol testing, BENZALKONIUM CHLORIDE
7. Clean and air-dry site SOLUTION (Zepharinchloride, 1:750) should be used for
8. Prepare equipment and put on gloves skin cleansing.
9. Reapply torniquet, uncap and inspect needle. ✓ Povidone iodine should be avoided as a cleansing agent
10. Ask patient to remake a fist, anchor vein, and insert because it interferes with several chemistry procedures.
needle. ✓ Pumping of the fist before venipuncture causes an
11. Establish blood flow, release torniquet and ask patient increase in plasma potassium, phosphate and lactate
to open fist concentrations.
12. Fill, remove, and mix tubes in order of draw ✓ The lowering of blood pH due to accumulation of lactate
13. Place gauze, withdraw needle, activate safety feature causes the IONIZED CALCIUM concentration to increase;
and apply pressure. this reverts to normal within 10 minutes after the
14. Discard needle and holder unit. torniquet is released.
15. Label tubes
16. Observe special handling instructions EVACUATED TUBES
17. Check patient’s arm and apply bandage Serum and Plasma Collection
18. Dispose used materials Plasma Value > No Plasma Value < Serum
19. Thank patient, remove gloves and sanitize hands. Serum Value, % difference Value, %
20. Transport specimen to the laboratory Calcium 0.9 Bilirubin Albumin 1.3
NOTES
Alkaline
✓ An intravenous (IV) infusion set, sometimes referred to Chloride 0.2 Cholesterol 1.6
phosphate
as butterfly because of the appearance of setup, maybe
Lactate Aspartate
used whenever the veins are fragile, small, or hard to 2.7 Creatinine 0.9
dehydrogenase aminotransferase
reach or find.
Total protein 4.0 Bicarbonate 1.8
✓ Sites adjacent to IV therapy should be avoided; if NOT
Creatinine kinase 2.1
possible, a site below the IV site should be sought. The
Glucose 5.1
initial sample drawn(5ml) should be discarded because
it is most like contaminated with IV fluid and only Phosphorus 7.0
subsequent samples are analyzed. Potassium 8.4
✓ Do not extract blood from patients while they are Sodium 0.1
receiving IV medications because these solutions may Urea 0.6
influence the analysis. Uric acid 0.2
✓ If a woman has undergone mastectomy, arm veins on
that side of the body should not be used because TUBE ADDITIVES
surgery may have caused lymphostasis (stop page of 1. Anticoagulants
flow of normal blood and lymph drainage through that ✓ prevents blood from clotting
site) affecting blood composition. ✓ EDTA, Citrates, Heparin, Oxalates
✓ If a woman has/had double mastectomy, blood should 2. Antiglycolyticagent
be drawn from the arm of the side on which the first ✓ prevents glycolysis, which can decrease glucose
procedure was done. concentration by up to 10 mg/dLper hour
✓ Sodium Flouride, Potassium Oxalate

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 Clinical Chemistry 1: Phlebotomy
3. Clot Activators
✓ are coagulation factors such as thrombin and
substances such as glass (silica) particles and
inert clays like diatomite (Celite)that enhance
clotting by providing more surface for platelet
activation. The clot activators in gel separator
4. Thixotropicgel
✓ separators are inert substances contained in or
near the bottom of certain tubes
✓ during centrifugation, the gel lodges between
the cells and the fluid, forming a physical
barrier that prevents the cells from
metabolizing substances in the serum or
plasma
Anticoagulants
Oxalate - it combines with calcium to form an
insoluble salt
Concentration: 1-2 mg/mL of blood (K+oxalate
0.01 ml of aq. 20% solution per mL of blood or 1
gt per 5mL of blood
Comments:
✓ Interferes with Na, K, and most BUN
(urease) tests
✓ Inhibits of LD, ACP, and AML
✓ Lithium oxalate- best oxalate to be used
for plasma uric acid
✓ Sodium oxalate- used routine lyin PT
tests
✓ Potassium oxalate–may cause variable
dilution of plasma due to water transport
from cells to the plasma (shrinks red
cells, thus, cannot be used to measure
hematocrit)
✓ Combined ammonium/potassium
oxalate does not have the same effect.
Citrate - it combines with calcium in a non-ionized
form
- buffered citrates stabilize plasma pH
- an insufficient blood volume (short
draw) leads to falsely increase clotting
time
- Inhibits AMYLASE activity
Concentration: 3.2% or 3.8% (0.105Mor0.129M)
in ratio of 1 part to 9 parts of blood.
Available as:
a. Light blue top tube
- containing 0.105 M or 0.129 M
(3.2%and3.8%) sodium citrate is
commonly used for coagulation
testing because it preserves the
labile coagulation factors
Ratio of anticoagulant is 1:9
b. Black top tubes
- also contain buffered sodium
citrate and are used for
Westergren ESR
Ratio of anticoagulant to blood is 1:4.
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- Ethylenediamine tetraacetic acid
- it combines with calcium in a process
called chelation
Preparation: Di-potassium (K2EDTA) in plastic
tubes and tri-potassium (K3EDTA) in glass tubes
Concentration: 1-2mg/mL of blood
Use in CC: Carcinoembryonic antigen (CEA), TDM,
and lead
Available as:
a. Lavender top tubes
✓ K2 EDTA- spray dried form
✓ K3EDTA- liquid form
b. Pink top tubes
✓ EDTA is spray-dried K2 EDTA)
✓ Used in immunohematology for
ABO grouping, Rh typing and
antibody screening.
✓ These tubes have a special
crossmatch label
c. White top tubes
✓ Contains EDTA and gel
✓ Mostly used for molecular
diagnostic testing
✓ Excess EDTA, which results
when tubes are underfilled, can
cause red blood cells to shrink
and thus, change blood count
cells.
Heparin - acts as antithrombin and
antithromboplastin, anti-FactorX, ideal
universal anticoagulant
- accelerates the action of antithrombin
III, neutralizing thrombin and preventing
the formation of fibrin
- Not suitable for nucleic acid testing
because it can be coextracted with DNA
and inhibits DNA polymerase in
polymerase chain reaction
Preparation: Sodium, lithium, potassium, and
ammonium salts
Concentration: 0.2mg/mL of blood
Uses: Tests for K+, NH3, carboxy/methemoglobin,
pH and blood gas and cytogenetic studies
Available as:
a. Sodium Heparin- injectable form used
for anticoagulant therapy
b. Lithium Heparin- used for most clinical
chemistry testing (measurements of
glucose, BUN, ionized calcium,
electrolytes and creatinine)
Fluoride - forms weakly dissociated calcium
components
- interferes with the measurement of Na,
K, and BUN (urease method)
- inhibits glycolytic enzymes enolase
(Mg2+ dependent enzyme) and glucose
oxidase
 Clinical Chemistry 1: Phlebotomy

- increases amylase activity Gold/Red Serum separator tube 5

- decreases ACP activity Red/red.
Serum tube, with/without clot
- used for glucose measurements since Orange/yellow, 5
activator, with/without gel
they contain preservative antiglycolytic Royal blue
agent Green Heparin tube with/without gel 8
- sodium fluoride prevents glycolysis for 3 Royal blue Sodium EDTA 8
days and iodoacetate for about 24 hours Lavender,
Concentration: 10 mg/mL of blood Pearl white,
EDTA tubes with/without gel 8
Pink/pink, Tan
(plastic)
Gray Glycolytic inhibitor 8
ACD for molecular studies and
Yellow (glass) 8
cell culture

ORDER OF THE DRAW

- a special sequence of tube collection that reduces the
risk of specimen contamination by microorganisms (e.g.,
blood cultures) and additive carry-over, which affects
some chemistry tests
- Most common tubes in the order of draw can be
remembered by recalling the phrase “Stop, Light Red,
Stay Put, Green Light, Go
- S (sterile), L (light blue), R (red), S (serum separator tube
or SST), P (plasma separator tube or PST), G (green), L
(lavender), G (gray)
CLSI ORDER OF DRAW
1. Sterile tube (e.g., blood culture)
2. Coagulation tube (e.g., blue top)
3. Serum tube with or without clot activator, with or
without gel (e.g., red top)
4. Heparin tube with or without gel separator (e.g., green
top)
5. EDTA tube with or without gel separator (e.g., lavender
top, pearl top)
6. Glycolytic inhibitor tube (e.g., gray top)
Recommended order of draw for multiple specimen collection
with tube color identification
Color Contents Inversions HANDLING OF SPECIMEN FOR ANALYSIS
Yellow Sterile media blood culture 8 Steps that are important in obtaining a valid specimen include
Royal blue No additive 0 1. Identification
No additive; discard tube if no 2. Preservation
Clear/Red 0 3. Separation and storage
royal blue used
Light blue Sodium citrate 3-4 4. Transport.

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 Clinical Chemistry 1: Phlebotomy

SPECIMEN IDENTIFICATION COMPLICATIONS OF VENIPUNCTURE

- Proper identification of the specimen must be Immediate a. Hemoconcentration- an increase in the
maintained at each step o the testing process Local number of formed elements in blood
- All labels should con form to the laboratory’s stated resulting from either a decrease or increase
requirements to facilitate proper processing of in plasma volume.
specimens. b. Failure of blood to enter the
PRESERVATION OF SPECIMEN syringe/vacutainer tube.
✓ The practitioner must ensure that specimens are • Excessive pull of plunger
collected into the correct container and are properly • Piercing the other pole of the vein
labelled • Trans fixation of vein
✓ Specimens must be properly treated both during • Incorrect bevel position
transport to the laboratory and from the time the • Absence of vacuum
serum, plasma, or cells have been separated until c. Hematoma
analysis • Failure to remove torniquet before
✓ Tests requiring 4 C from the time blood is drawn until withdrawing the needle
the specimens are analyzed: • Failure to have the needle
a. Ammonia completely in the vein
b. Lactate • Repeated punctures of the vein
c. Certain Hormone tests (gasrtin, renin activity)
• Failure to apply pressure to the
d. Blood Gas Determination (PCO2, PO2, and
wound for a sufficient length of time.
blood pH)
d. Circulatory failure
✓ Tests requiring protection from daylight and fluorescent
e. Syncope (fainting)
light (prevent photodegradation):
Late local a. Thrombosis –an abnormal vascular
a. Bilirubin
condition in which thrombus develops within
b. Carotene
a blood vessel of the body.
c. Methotrexate
b. Thrombophlebitis –inflammation of a vein
SEPARATION AND STORAGE OF SPECIMENS
often accompanied byua clot as a result of
✓ Plasma or serum should be separated from cells, either
trauma to vessel wall.
by removal of the liquid to an aliquot tube or by
Late General a. Serum Hepatitis
migration of a gel barrier during centrifugation, as soon
b. AIDS
as possible and certainly within 2 hours
✓ If it is impossible to centrifuge a blood specimen within
SKIN PUNCTURE
2 hours, the specimen should be held at room
- mixture of blood coming from arterioles, venules, and
temperature rather than at 4°C to decrease hemolysis.
capillaries
✓ For most plasma samples used or molecular diagnostics,
- preferred method for pediatric patients.
the plasma should be removed from the primary tube
Sites
promptly after centrifugation and held at −20 °C in a
1. Infants: Plantar heel surface
freezer capable of maintaining this temperature.
2. Children: plantar surface of the big toe, palmar surface
TRANSPORT OF SPECIMENS
of the last digit of the second, third, or fourth fingers;
✓ Shipping of Specimen from one laboratory to another.
lateral side of the finger adjacent to the nail, earlobe
✓ Sending out of samples to a Reference laboratory for
NOTES:
Confirmation.
✓ If using a lancet the blade should be no longer than 2.4
10 COMMON ERRORS IN SPECIMEN COLLECTION
mm to avoid injury to the calcaneus (heel bone).
1. Misidentification of patient
✓ Skin puncture is useful in adults with:
2. Mislabeling of specimen
a. Extreme obesity
3. Short draws / wrong anticoagulant to blood ratio
b. Severe burns
4. Mixing problems / clots
c. Thrombotic tendencies
5. Wrong tubes / wrong anticoagulant
✓ Skin puncture is often preferred in geriatric patients
6. Hemolysis / Lipemia
because skin is thinner and less elastic; thus a hematoma
7. Hemoconcentration from prolonged torniquet time
is more likely to occur from a venipuncture.
8. Exposure to light / Extreme temperatures
✓ In newborns, skin puncture is frequently used to collect
9. Improperly timed specimens / delayed delivery to
a sample for bilirubin testing
laboratory
✓ In older pediatric population, earlobe blood is available,
10. Processing errors: Incomplete centrifugation, incorrect
but in neonates and infants, it is impractical to sample
log-in, incorrect storage
THE earlobe, therefore the heel is more often used.

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 Clinical Chemistry 1: Phlebotomy

✓ A deep heel prick is made at the distal edge of the REASONS FOR RAPID SEPARATION OF BLOOD
calcaneal protuberance following a 5-10 minute 1. To prevent glycolysis
exposure period to prewarmed water. 2. To prevent lipolysis
✓ The best method for blood gas collection in the newborn 3. Certain substances are very unstable
still remains the indwelling umbilical artery catheter. 4. To prevent shift of electrolytes
Order of draw 5. To prevent hemolysis
1. Blood Gases
2. Slide/Smear
3. EDTA Tubes
4. Other additive mini containers
5. Serum containers, mini containers
SERUM
- defined as the liquid portion of blood that remains after
coagulation has occurred
- it is the specimen of choice for many analyses, including
viral screening and protein electrophoresis
- samples are collected into tubes with no additive or with
a clot activator and must be allowed to complete the
coagulation process before further processing
- does not contain fibrinogen and some potassium is
released from platelets
PLASMA
- defined as the noncellular component or anti
coagulated whole blood
- it contains clotting factors
Differences of Plasma and Serum
Plasma Serum
More protein
Protein Less protein (fibrin)
(fibrinogen)
Clarity Less clear Clearer
Absent (appears to
Lipemia clearing be coprecipitated
Present
factor during clotting
process)
Higher (platelets
Potassium lower
release K+)

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