Phlebotomy – the act of drawing or removing blood from circulatory system thru a puncture to obtain a

specimen for analysis and diagnosis

Venipuncture – the puncture of a vein for surgical or therapeutic purposes or for collecting blood
specimens for analysis

I. VENIPUNCTURE

BLOOD COLLECTION EQUIPMENT

1. Blood-drawing station
2. Phlebotomy chair
3. Handheld phlebotomy equipment carriers & phlebotomy cart
4. Gauze pads or cotton balls
5. Bandages
6. Glass microscope slides (1 by 3-in)
7. Tourniquet
 Device applied or tied around patient’s arm to restrict blood flow
 used to provide a barrier against venous blood flow to help locate a vein.
 should be applied 3 to 4 inches (7.5 to 10 cm) above the venipuncture site and
left on for no longer than 1 minute before the venipuncture is performed to
prevent hemoconcentration.

8. Collection Tubes
 The most common means of collecting blood specimens is through the use of an
evacuated tube system. The system includes an evacuated tube, which can be
either plastic or glass; a needle; and an adapter that is used to secure the needle
and the tube.
 When the needle is inserted into a vein and a tube is inserted into the holder, the
back of the needle pierces the stopper, allowing the vacuum pressure in the tube
to automatically draw blood into the tube.
 For safety, OSHA recommends the use of plastic tubes whenever possible. Most
glass tubes are coated with silicone to help decrease the possibility of hemolysis
and to prevent blood from adhering to the sides of the tube.

9. Needles
 The gauge number of a needle is inversely related to the bore size: the smaller the
gauge number, the larger the bore.
 Needles for drawing blood range from 19 to 23 gauge.
 The most common needle size for adult venipuncture is 21 gauge with a length of 1 –
1.5 inch The advantage of using a 1-inch needle is that it provides better control
during venipuncture.
 TYPES OF NEEDLES:
o Multisample
o Hypodermic
o Winged Blood Collection Set - Butterfly
 useful in collecting specimens from children or other patients from
whom it is difficult to draw blood. They also have sheathing devices
to minimize the risk of needle stick injury.
 PARTS OF NEEDLE:
o Bevel, shaft, hub and lumen
 NEEDLE GAUGE
o 16 – white
o 18 – pink
o 19 – cream
o 20 – yellow
o 21 – deep green
 o 22 – black
o 23 – deep blue (combination w/ butterfly collection)
o 24 – medium purple
o 25 – orange (intermuscular injection)
o 26 – brown
o 27 – gray (PPD skin test)
o 29 – red
o 18 – 16 = IV infusion

10. Needle Holders

 Needle holders are made to fit a specific manufacturer’s needles and tubes and should not
be interchanged.
 The holders are disposable and must be discarded after a single use with the needle still
attached as required by OSHA

11. Syringes
 consists of a barrel, graduated in milliliters, and a plunger.
 useful in drawing blood from pediatric, geriatric, or other patients with tiny, fragile, or
“rolling” veins that would not be able to withstand the vacuum pressure from evacuated
tubes.
 With a syringe, the amount of pressure exerted is controlled by the phlebotomist by
slowly pulling back the plunger.
 Syringes may also be used with winged infusion sets. If only one tube of blood is needed,
the phlebotomist fills the syringe barrel with blood, removes the needle from the arm,
activates the needle safety device, removes and discards the needle in a sharp container,
and attaches the hub of the syringe to a transfer device to transfer the blood into an
evacuated tube.
 With this system, the butterfly needle tubing branches into a Y shape and attaches to the
syringe on one side and an evacuated tube in a holder on the other side. Clamps in the
tubing control the flow of blood from the arm to the syringe and then from the syringe to
the evacuated tube.
 To prevent hemolysis when using transfer devices, only the tube’s vacuum (and not the
plunger) should be used to transfer the blood from the syringe into the evacuated tube.

ADDITIVES
1. Clot activators
 Blood specimens for serum testing must first be allowed to clot for 30 to 60 minutes prior
to centrifugation and removal of the serum.
 A clot activator accelerates the clotting process and decreases the specimen preparation
time.
 Examples include glass or silica particles (activates factor XII in the coagulation
pathway) and thrombin (an activated coagulation factor that converts fibrinogen to fibrin)

2. Anticoagulants
 prevents blood from clotting/ coagulation & allow separation of blood into cellular &
liquid (plasma) components
 Ethylenediaminetetraacetic acid (EDTA), citrate, and oxalate remove calcium needed for
clotting by forming insoluble calcium salts.
 Heparin prevents clotting by binding to antithrombin in the plasma and inhibiting
thrombin and activated coagulation factor X
 Tubes with anticoagulant are either tested as whole blood or are centrifuged to yield
plasma.

1. POTASSIUM OXALATE / SODIUM FLUORIDE

o Gray stoppered-tubes contain oxalates in combination with a weak antiglycolytic
agent, sodium fluoride
o Potassium oxalate - works by precipitating out the calcium in the blood and
therefore stopping the coagulation cascade
 o Sodium fluoride – its primary function is not as an anticoagulant but as glycolytic
inhibitor. Fluoride preserves the glucose in the blood sample by inhibiting the
enzymes involved in breakdown of glucose (glycolysis)
o Precipitates calcium; inhibits glycolysis

2. SODIUM CITRATE
o Light blue-stoppered tubes contain the anticoagulant sodium citrate
o Prevents the coagulation by binding calcium in nonionized form
o 3.8% sodium citrate (0.129 M) or 3.2% (0.105 M) concentrations
o If a tube is only half-filled, ratio will be off and the results will be invalid
o Three tests using the light blue-stoppered tube to detect clotting problems are:
PT, aPTT and fibrinogen assay
o Citrate containing citrate, theophylline, adenosine, dipyridamole (CTAD) is a
light blue-stoppered tube that is used for platelet function assays and some
routine coagulation determination
o Anticoagulant of choice for coagulation and platelet function tests, also used for
ESR (erythrocyte sedimentation rate test)
o Came in a liquid form as 3.8% tri-sodium citrate
o For coagulation testing, ratio of 9 volumes of blood to one volume of
anticoagulant (9 volumes blood: 1 volume anticoagulant) is very important, as
variation from this ratio may cause errors
o For ESR, 4 volumes of blood to one volume of anticoagulant is used (4:1)

3. SOLUTIONS A and B (ACD)

o Alternate yellow-stoppered tubes that are labeled as solution A or solution B.
These two types of tubes contain variations of a mixture of trisodium citrate,
citric acid and dextrose
o Used for DNA and paternity testing, human leukocyte antigen (HLA)
phenotyping and some immunohematology studies

4. SODIUM POLYANETHOL SULFONATE (SPS)

o A yellow-stoppered tube that does not contain citrate
o Used for collecting blood culture samples
o Main function: to allow bacteria to grow so they can be cultured
o Inhibits phagocytosis of the bacteria by the white blood cells (WBCs)
o Inhibits serum complement which would destroy the bacteria
o Inhibits certain antibiotics in case patient is already on an antibiotic

5. EDTA
o Lavender tubes contain anticoagulant EDTA
o Binds calcium to prevent coagulant
o Is in the form of tripotassium EDTA (K3 or sequestrene) and dipotassium EDTA
(K2 or versine)
o Tripotassium EDTA (K3) is used in glass tubes and is in the liquid form
o Dipotassium EDA (K2) is in plastic tubes in spray-dried powdered
 Recommened because it preserves the cell morphology for complete
blood cell counts (CBCs) and differential blood smears and provides
stable microhematocrit results
o Optimal anticoagulant concentration of 1.5 mg/mL
o can be found in three salt forms: Tri-potassium EDTA, Di-sodium EDTA and
Di-lithium EDTA
o Acts as chelating / removing ionized calcium (Calcium is required for blood to
clot, so when it is removed, blood will not clot)
o Commonly used anticoagulant in hematology section & it is the anticoagulant of
choice for CBC

6. HEPARIN
o Green tubes contain heparin which stops the coagulation by inhibiting the
conversion of prothrombin to thrombin and thus the following stages that lead to
a clot
o No fibrin is formed to cause clot
 o Naturally occurring substance that is present in most of our tissues but at low
levels
o Anticoagulant of choice for pH determinations, electrolyte studies and arterial
blood gas
o Comes in three forms: lithium heparin, sodium heparin and ammonium heparin
o Acceptable for blood samples that may be stored for more than 48 hours before
testing
o Optimal anticoagulant concentration of 15 – 12 U / mL
o An acid mucopolysaccharide, it acts by complexing with ant-thrombin to prevent
blood clotting (antithrombin is one of the natural/physiological inhibitors of
blood coagulation which is found in vivo)
o Preferred anticoagulant for osmotic fragility test
o Also used in capillary tubes for spun hematocrit (HCT)
o Also used for L.E cell preparation (L.E = lupus erythromatosus)
o Found in basophil and mast cell granules
o Used therapeutically as an in vivo anticoagulant

7. EDTA WHITE-STOPPERED TUBES

o Also known as pearl top contains EDTA as an anticoagulant and also gel to
separate the plasma from cells
o Helpful in working with human immunodeficiency virus (HIV) positive patients

8. TRACE ELEMENT TUBES

o Royal blue-stoppered tube is used for trace element studies – that is analysis of
such trace elements as lead, zinc, arsenic or copper
o Normal serum or plasma blood tubes cannot be used
o Patient contacts these elements through occupational or environmental exposure
o If blood was drawn into normal tubes, trace elements would leach out of the glass
and rubber stopper to falsely elevate the results

3. Antiglycolytic Agent
 An antiglycolytic agent inhibits the metabolism of glucose by blood cells. Such inhibition
may be necessary if testing for the glucose level is delayed.
 The most commonly used antiglycolytic agent is sodium fluoride.
 Tubes containing sodium fluoride alone yield serum.
 Tubes containing sodium fluoride and an anticoagulant (such as EDTA or oxalate) yield
plasma.
 Anticoagulated blood can be centrifuged immediately to obtain plasma for testing, thus
decreasing the specimen preparation time.

4. Separator Gel
 Separator gel is an inert material that undergoes a temporary change in viscosity during
the centrifugation process; this enables it to serve as a separation barrier between the
liquid (serum or plasma) and cells.
 Because this gel may interfere with some testing, serum or plasma from these tubes
cannot be used with certain instruments or for blood bank procedures.
 BD Vacutainer® Venous Blood Collection
TUBE GUIDE:

BD BD Additive Inversion Laboratory Use

Vacutainer® Vacutainer® s
Tubes with Tubes with
BD Conventional
Hemogard™ Stopper
Closure
GOLD RED/GRAY Clot activator and 5 a. serum determinations in chemistry
(serum) gel for serum b. routine blood donor screening and diagnostic
separation testing of serum for infectious disease.

Tube inversions ensure mixing of clot activator with

blood. Blood clotting time: 30 minutes

o ANTICOAGULANT: none
o MODE OF ACTION: blood clots and the
serum is separated after centrifugation
o TEST: chemistry, serology, blood bank
(crossmatching
o Has thixotropic are semipermeable gel
(barrier between red blood cells and serum)
o Specific gravity:
o red blood cell is 1.09 – 1.010
o Plasma is 1.03
o Thixotropic gel is 1.04 – 1.05

LIGHT GREEN/GRAY Lithium heparin 8 a. plasma determinations in chemistry

GREEN and gel for plasma
(plasma) separation Tube inversions ensure mixing of anticoagulant
(heparin) with blood to prevent clotting.

 Additive: Lithium heparin; plasma separation

gel
 Type: Anticoagulant separator tube
 Used For: Obtaining plasma

RED (serum) RED • Silicone coated 0 a. serum determinations in chemistry

(glass) b. routine blood donor screening and diagnostic
• Clot activator, 5 testing of serum for infectious disease
Silicone coated
(plastic) Tube inversions ensure mixing of clot activator with
blood. Blood clotting time: 60 minutes.

 Additive: None (glass tubes), Clot activator

(plastic tubes)
 Used for: Obtaining serum; chemistries
 Type: clot tube
 Common Tests: ABO/RH, Crossmatch,
Digoxin, Tegretol, Dilantin/Phenytoin
o chemistry, serology, blood bank

ORANGE Thrombin-based 5-6 a. stat serum determinations in chemistry.

(serum) clot activator with
gel for serum Tube inversions ensure mixing of clot activator with
separation blood. Blood clotting time: 5 minutes
ORANGE Thrombin-based 8 a. stat serum determinations in chemistry
(serum) clot activator
Tube inversions ensure mixing of clot activator with
 blood. Blood clotting time: 5 minutes

 Additive: Thrombin-based clot activator;

separator gel (in some)
 Type: Clot Tube
 Used for: Obtaining serum

ROYAL • Clot activator 8 b. For trace-element, toxicology, and nutritional-

BLUE (plastic serum) chemistry determinations.
(plasma) c. Special stopper formulation provides low levels
• K2EDTA of trace elements (see package insert).
(plastic)
Tube inversions ensure mixing of either clot activator
or anticoagulant (EDTA) with blood.

 Additive: K2EDTA or Clot Activator

 Type: Low trace-element testing
 Used for: Trace metal analysis, toxicology
and drug level testing
 Notes: Be sure to use a tube with the proper
additive; Royal Blue top indicates that the
tube is low in trace-elements, not which
additive is present

GREEN GREEN • Sodium heparin • 8 a. plasma determinations in chemistry.

(plasma) Lithium heparin
Tube inversions ensure mixing of anticoagulant
(heparin) with blood to prevent clotting.

 Additive: Sodium heparin (lithium level) or

Lithium Heparin (ammonia level)
 Type: Anticoagulant
 Used For: Plasma chemistry; electrolytes
 Common Lab Tests: Electrolytes, STAT
BMP, CMP, Glucose, Potassium, Troponin,
Lactate (on ice), ammonia, HLA typing

GRAY GRAY • Potassium 8 b. For glucose determinations.

(plasma) oxalate/ sodium c. Oxalate and EDTA anticoagulants will give
fluoride plasma samples.
• Sodium d. Sodium fluoride is the antiglycolytic agent.
fluoride/Na2
EDTA • Sodium Tube inversions ensure proper mixing of additive
fluoride (serum with blood
tube)
 Additive: Sodium Fluoride & Potassium
Oxalate OR, Sodium Fluoride & Na2 EDTA
OR Sodium Fluoride
 Type: Anticoagulant
 Used for: Plasma glucose testing
 Notes: Preserves glucose for 1-5 days.
Hemolysis in partially full tubes (full draw)

TAN (plasma) K2EDTA (plastic) 8 a. For lead determinations. This tube is certified to
contain less than .01 g/mL(ppm) lead.

Tube inversions prevent clotting.

 Additive: K2EDTA
 Type: Antocoagulant
 Used For: Testing lead levels
 Notes: Tan stopper indicates that the tube is
certified to be low in trace lead content
 YELLOW • Sodium 8 b. SPS for blood culture specimen collections in
polyanethol microbiology.
sulfonate (SPS)
 Additive: Sodium polyanethol
• Acid citrate sulfonate (SPS)
dextrose additives  Type: Anticoagulant, Preservative
(ACD):
Solution A c. ACD for use in blood bank studies, HLA
22.0 g/L trisodium phenotyping, and DNA and paternity testing.
citrate
8.0 g/L citric acid  Additive: Acid, citrate, & dextrose
24.5 g/L dextrose  Type: Preserves red blood cells
Solution B  Used For: Genetic DNA & paternity
13.2 g/L trisodium Testing, blood bank studies, HLA
citrate phenotyping
4.8 g/L citric acid
14.7 g/L dextrose Tube inversions ensure mixing of anticoagulant with
blood to prevent clotting.
LAVENDER LAVENDER • Liquid K3EDTA 8 a. K2EDTA and K3EDTA for whole blood
(whole blood) (glass) hematology determinations.
• Spray-coated b. K2EDTA may be used for routine
K2EDTA (plastic) immunohematology testing, and blood donor
screening.

Tube inversions ensure mixing of anticoagulant

(EDTA) with blood to prevent clotting.

 Additive: K3EDTA (Glass) or spray-coated

K2EDTA (Plastic)
 Type: Anticoagulant
 Used For: Hematology Testing (CBC)
 Notes: Prevents artifacts for up to 12 hours,
partially filled tube damages cells (full draw)
 Common Lab Tests: complete blood count
(CBC), white blood cell (WBC), hematocrit
(Hct), erythrocyte sedimentation rate (ESR),
Platelet Count, Ammonia (on ice),
hemoglobin A1c (HgbA1C), red blood cell
(RBC), hemoglobin (Hgb), Differential count,
cross matching,

WHITE K2EDTA and gel 8 a. For use in molecular diagnostic test methods
for plasma (such as, but not limited to, polymerase chain
separation reaction [PCR] and/or branched DNA [bDNA]
amplification techniques.)

Tube inversions ensure mixing of anticoagulant

(EDTA) with blood to prevent clotting.

 Additive: K2EDTA; plasma separation gel

 Type: Anticoagulant
 Used For: Molecular diagnostics
 Notes: Plasma based testing performed on
proteins

PINK (whole PINK Spray-coated 8 b. For whole blood hematology determinations.

blood) K2EDTA (plastic) c. May be used for routine immunohematology
testing and blood donor screening.
d. Designed with special cross-match label for
patient information required by the AABB.

Tube inversions prevent clotting

 Additive: spray-coated K2EDTA

 Type: Anticoagulant
  Used for: Immunohematology
 Notes: Used in blood typing

LIGHT BLUE LIGHT BLUE • Buffered sodium 3-4 a. For coagulation determinations.
(plasma) citrate 0.105 M (- b. CTAD for selected platelet function assays and
3.2%) glass 0.109 routine coagulation determination.
M (3.2%) plastic
• Citrate, Tube inversions ensure mixing of anticoagulant
theophylline, (citrate) to prevent clotting.
adenosine,
dipyridamole  Additive: Sodium Citrate
(CTAD)  Type: Reversible Anticoagulant
 Used For: Coagulation testing; plasma
 NOTE: tube must be filled 100%
 Common Lab Tests: prothrombin time (PT)
or thrombin clotting time (TCT), partial
prothrombin time (PTT), Fibrinogen assay,
prothrombin time/ international normalized
ratio (PT/INR)

CLEAR RED/LIGHT • None (plastic) 0 For use as a discard tube or secondary specimen tube.
GRAY
RED / GRAY  Additive: Clot activator; separation gel
TIGER TOP /  Used for: Obtaining serum
GOLD (SST)  Type: clot tube
 Common Tests:
o basic metabolic panel (BMP; sodium,
potassium, chloride, carbon dioxide
CO2, blood urea nitrogen BUN,
calcium & creatinine)
o comprehensive metabolic panel
(CMP; glucose, BUN, creatinine,
sodium, potassium, chloride, calcium,
carbon dioxide, albumin, total
protein, alkaline phosphatase,
aspartate aminotransferase AST,
alanine aminotransferase ALT & total
bilirubin)
o Bilirubin, Thyroid Testing, Hormone
Levels, General Chemistry, creatine
kinase-MB (CKMB), human
chorionic gonadotropin (hCG), PSA
CK

BLACK  Additive: Varies; often sodium citrate

(whole blood)  Type: Anticoagulant
 Used for: Erythrocyte Sedimentation Rate
 Notes: Also called a Westerngren
Sedimentation Rate (full draw)

ADDITIONAL NOTES FOR TUBES:

1. Red Top Tube

 anticoagulant: none; uses/tests: blood chemistries, serology or any test that requires serum; yields
serum after centrifugation

2. Serum Separator Tube; Tiger Top Tube

 anticoagulant: none but does contain a clot activator, polymer gel, and silicone plug to keep
serum and clotted blood/cells separated; uses/tests: blood chemistries, serology or any test that
requires serum; yields serum after centrifugation
 3. Lavender/Purple Top Tube
 anticoagulant: ethylenediaminetetraacetic Acid (EDTA); uses/tests: general hematology, CBC
blood films/ smears; yields plasma after centrifugation

4. Blue Top Tube

 anticoagulant: sodium citrate; uses/tests: coagulation/clotting studies or tests; yields plasma after
centrifugation

5. Green Top Tube

 anticoagulant: lithium heparin; uses/tests: pH levels, lead determinants, or some blood
chemistries; yields plasma after centifugation

6. Gray Top Tube

 anticoagulant: potassium oxalate and/or sodium fluoride; uses/tests: glucose determinants; yields
plasma after centrifugation

Selection of a Vein for Routine Venipuncture

 The superficial veins of the antecubital fossa (bend in the elbow) are the most common
sites for venipuncture.
 There are two anatomical patterns of veins in the antecubital fossa.
o In the “H” pattern, the three veins that are used:
1. the median cubital vein, which connects the basilic and cephalic veins
in the antecubital fossa;
2. the cephalic vein, located on the outside (lateral) aspect of the
antecubital fossa on the thumb side of the hand; and
3. the basilic vein, located on the inside (medial) aspect of the antecubital
fossa.
o In the “M” pattern, the order of preference is the
1. median vein,
2. accessory cephalic vein, and
3. the basilic vein.
 The cephalic and basilic veins should only be used if the median
cubital or median veins are not prominent after checking both
arms.
 The basilic vein is the last choice due to the increased risk of
injury to the median nerve and/or accidental puncture of the
brachial artery, both located in close proximity to the basilic
vein.

VENIPUNCTURE PROCEDURE

 The phlebotomist uses standard precautions, which include washing hands and applying
gloves at the beginning of the procedure and removing gloves and washing hands at the
end of the procedure. The Clinical and Laboratory Standards Institute (CLSI)
recommends the following steps:

1. Prepare the accession (test request) order.

TEST REQUEST FORM SHOULD CONTAIN:
o Patient’s complete name
o Age
o Date of birth
o Patient identification number
 o Type of test to be collected
o Date and time the sample is to be obtained
o Department or location of patient
o Clinical impression/diagnosis
o Physician’s name

2. identify the patient by having the patient verbally state his or her full name and
confirm patient’s unique identification number, address, and/ or birth date.
Ensure the same information is on the request form.

 PROPER PATIENT IDENTIFICATION:

o CONSCIOUS PATIENTS (out-patient):

 ask patient to give their full name and spell their last name
 Compare the information on the request form
o CONSCIOUS PATIENTS (in-patient):
 ask patient to give their full name and spell their last name
 Compare the information on the request form and on their
identification bracelet
o SLEEPING PATIEN:
 Awaken a sleeping patient before attempting venipuncture
 If the patient is already awake, do the same steps as
conscious patients
o SEMI-CONSCIOUS OR COMATOSE PATIENTS:
 Ask the watcher (or nurse, if no watcher is around) to
identify the patient
 Compare the information on the identification bracelet and
request form
o TOO YOUNG, MENTALLY INCOMPETENT OR DO NOT SPEAK
THE LANGUAGE OF A PHLEBOTOMIST:
 Ask the watcher (or nurse, if no watcher is around) to
identify the patient
 Compare the information on the identification bracelet and
request form
o UNIDENTIFIED EMERGENCY PATIENT
 Upon admission, a temporary identification number will be
assigned to the patient. Used this ID number on all tests
 When a permanent number or when the patient has
already been identified, cross-reference it with temporary
number

3. Sanitize hands.
4. Verify that any dietary restrictions have been met (e.g., fasting, if appropriate)
and check for latex sensitivity.
5. Assemble supplies and appropriate tubes for the requested tests. Verify
paperwork and tube selection.
6. Reassure and position the patient.
7. If necessary to help locate a vein, request that the patient clench his or her fist.
8. Apply the tourniquet and select an appropriate venipuncture site
9. Put on gloves.
10. Cleanse the venipuncture site with 70% isopropyl alcohol using concentric
circles from the inside to outside. Allow skin to air-dry.
11. Inspect the equipment and needle tip for burrs and bends.
12. Perform the venipuncture by anchoring the vein with the thumb 1 to 2 inches
below the site and inserting the needle, bevel up, with an angle less than 30
degrees between the needle and the skin. Collect tubes using the correct order of
draw, and invert each tube containing any additive immediately after collection.

SITES TO BE AVOIDED:
  Burns, scars, or tattoos
 Damaged veins
 Edema
o Are swollen because of fluid in the tissue; veins are not prominent;
toruniquet will be ineffective due to the swelling
 Hematoma
o Samples collected may cause erroneous test results
o If another vein site is not available, sample is collected distal from
hematoma
 Mastectomy
o Because of the potential for harm to the patient due to
lymphostasis, arm on the side of a mastectomy should be avoided
o If the patient has double mastectomy, physician should be
consulted prior to drawing blood. Usually the draw will be
performed on the side opposite the newest mastectomy
 IV-line, cannula, fistula
o Should have samples collected from the opposite arm
o Blood should never be collected above the running IV
o Areas of scarring are also to be avoided because of the possible
injury to the patient pr excessive pain

13. Release and remove the tourniquet as soon as blood flow is established
14. Ensure that the patient’s hand is open.
15. Place gauze lightly over the puncture site without pressing down.
16. After the last tube has been released from the back of the multisample needle,
remove the needle and activate the safety device according to the manufacturer’s
directions.
17. Apply direct pressure to the puncture site using a clean gauze pad.
18. Bandage the venipuncture site after checking to ensure that bleeding has stopped.
19. If a syringe has been used, fill the evacuated tubes using a syringe transfer
device.
20. Dispose of the puncture equipment and other biohazardous waste.
21. Label the tubes with the correct information. The minimal amount of information
that must be on each tube is as follows:
a. Patient’s full name
b. Patient’s unique identification number
c. Date of collection
d. Time of collection (military time)
e. Collector’s initials or code number

Note: Compare the labeled tube with the patient’s identification bracelet or
have the patient verify that the information on the labeled tube is correct
whenever possible.

22. Carry out any special handling requirements (e.g., chilling or protecting from
light).
23. Cancel any phlebotomy-related dietary restrictions and thank the patient.
24. Send the properly labeled specimens to the laboratory.

 The most crucial step in the process is patient identification.

ORDER OF DRAW FOR VENIPUNCTURE

1. Blood culture tube or vials (yellow stopper)
2. Coagulation tube (light blue stopper)
3. Serum tube with or without activator (red, gold, red-gray marbled, orange, or yellow-gray
stopper)
4. Heparin tube (green or light green stopper)
5. EDTA tube (lavender or pink stopper)
 6. Sodium fluoride with or without EDTA or oxalate (gray stopper)

PROBLEMS ENCOUNTERED IN VENIPUNCTURE:

1. CARDIAC ARREST
o Patient falls into unconsciousness, no pulse or respiration, dilated eyes and pale
skin
o Immediate CPR

2. CONTINUED BLEEDING
o Some patients take more than 5 minutes for the site to stop bleeding
o Continue to wrap an elastic gauze around the arm with pad
o Leave it on for 15 minutes or until the bleeding stops

3. HEMATOMA
o The puncture should not be too deep to pass through the top and bottom walls of
the vein (through and through)
o Discontinue venipuncture and apply heavy pressure

4. SKIN ALLERGIES
o Some patients are allergic to latex, tape or iodine
o Use hypoallergenic tape and non-latex elastic wrap

UNUSUAL BLOOD SAMPLE:

1. ICTERIC SAMPLE
o Serum/plasma that contains large amount of bilirubin
o Patient presents with jaundice

2. LIPEMIC SAMPLE
o Serum/plasma contains large amount of fats and lipids
o May be due to patient not fasting

3. HEMOLYZED SAMPLE
o serum/plasma contaminated eith RBC contents

CAUSES OF HEMOLYSIS:
A. Drawing from hematoma
B. Rupturing of RBCs by using a needle that it is too small
C. Alcohol on the site of venipuncture that entered the blood sample
D. Pulling the plunger too forcibly
E. Fast drip/expelling blood vigorously as it is transferred to the tube
F. Redirecting
G. Mixing tubes vigorously

POSSIBLE CAUSES FOR FAILED VENIPUNCTURE

1. vacuum in tube is not working
2. Bevel avainst the vein wall
3. Bevel inserted too far
4. Needle partially inserted
5. Needle slipped beside the vein
6. Collapsed vein
7. Undetermined needle position
TECHNIQUES TO ENHANCE VEIN AND RECOVER A FAILED VENIPUNCTURE

 retie the tourniquet

 use a blood pressure cuff in place of a tourniquet
 massage the arm or warm the location
 lower the patient's arm
 reseat the tube holder
 use a different tube
 place your finger below the venipunctrue site and stretch the vein slightly
 pull back or advance the needle slightly
 rotate the needle onr quarter to one half turn. Make sure to pull a little backward before
redirecting
 venipuncture attempts should be up to 2 tries only. Ask someone else to do it (endorse
to another staff)

TEN MOST COMMON ERRORS IN SPECIMEN COLLECTION

1. Misidentification of patient
2. Mislabeling of specimen
3. Shortd raws/wrong AC/blood ratio
4. Mixing problems/clots
5. Hemolysis/lipemia
6. hemoconcentration from prolonged tourniquet time
7. Exposure to light/extreme temperature
8. Improperly timed specimen / delayed delivery to the laboratory
9. Processing errors: incomplete centrifugation, improper storage

II. SKIN PUNCTURE

 is the technique of choice to obtain a blood specimen from newborns and pediatric
patients.
 In adults, skin puncture may be used in patients who are severely burned and whose veins
are being reserved for therapeutic purposes; in patients who are extremely obese; and in
elderly patients with fragile veins.
 Blood obtained from skin puncture is a mixture of blood from venules, arterioles,
capillaries, and interstitial and intracellular fluids
 A mixture of arterial blood, venous blood and tissue fluid
 Contains interstitial and intracellular fluids

Parameter Characteristic of Capillary Blood (in

comparison to Venous Blood)
Packed Cell Volume Slightly Higher
RBC Count Slightly Higher
Hemoglobin Slightly Higher
WBC Count 15 – 20% Higher
Monocytes 12% Higher
Platelet Count Lesser

COLLECTION SITE

 site of choice for skin puncture in infants under 1 year of age is the lateral (outside) or
medial (inside) plantar (bottom) surface of the heel
 In children older than 1 year of age and in adults, the palmar surface of the distal portion
of the third (middle) or fourth (ring) finger on the nondominant hand may be used.
 The puncture on the finger should be made perpendicular to the fingerprint lines
 Fingers of infants should not be punctured because of the risk of serious bone injury.
 Fleshy portion of the last phalanx of the 3rd or 4th finger of the non – dominant hand
 Lateral plantar heel surface (newborn)
PRECAUTIONS WITH SKIN PUNCTURE

 The finger or heel must be securely immobilized.

 Heel punctures in infants should not be made more than 2 mm deep because of the risk of
bone injury and possible infection (osteomyelitis)
 In premature infants, a puncture device that makes an incision with even less depth is
preferred.
 The phlebotomist should not puncture an area that is swollen, bruised, infected, or
already has been punctured. In addition phlebotomists should not perform skin puncture
in patients with edema, dehydration, or poor peripheral circulation because specimen
integrity and test accuracy may be compromised.
 The first drop of blood should be wiped away with a clean gauze pad to prevent
contamination of the specimen with tissue fluid and to facilitate the free flow of blood

EQUIPMENT FOR SKIN PUNCTURE

 sterile lancets that puncture or sterile blades that make a small incision in the skin.
 Containers for collecting blood from skin puncture include capillary tubes and
microcollection tubes. The order of draw, however, is different for microcollection tubes
 The EDTA microcollection tube should be filled first to ensure adequate volume and
accurate hematology results, especially for platelets, which tend to aggregate at the site of
puncture.
 Disinfectant
a.) 70% Isopropyl Alcohol - BEST
(Iodine tincture preparations are not recommended for disinfecting skin
puncture sites because they can falsely elevate potassium, uric acid,
phosphorus)
 Puncturing Device (no longer used)
a.) Hagedorn Needle (a large needle with cord at one end)
b.) Glover’s Needle (3 cornered short needle)
 Appropriate Capillary Tubes
a.) Red Tip (heparinized)
b.) Blue Tip (no anticoagulant)
 Micro – containers

SKIN PUNCTURE PROCEDURE

 The phlebotomist uses standard precautions that include washing hands and applying
gloves at the beginning of the procedure and removing gloves and washing hands at the
end of the procedure. CLSI recommends the following steps:
1. Prepare the accession (test request) order.
2. Greet the patient (and parents); identify the patient by having the patient (or parent in
the case of a child) verbally state his or her full name and confirm with patient’s
identification number, address, and/or birth date. Ensure that the same information is
on the requisition form.
3. Position the patient and the parents (or individual designated to hold an infant or
small child) as necessary.
4. Verify that any dietary restrictions have been met (e.g., fasting), and check for latex
sensitivity.
5. Wash hands and put on gloves.
6. Assemble supplies and appropriate tubes for the requested tests. Check paperwork
and tube selection.
7. Select the puncture site.
8. Warm the puncture site.
9. Cleanse the puncture site with 70% isopropyl alcohol using concentric circles,
working from the inside to outside. Allow skin to air-dry.
10. Open and inspect the sterile disposable puncture device, and perform the puncture
while firmly holding the heel or finger. Discard the device in the appropriate sharps
container.
11. Wipe away the first drop of blood with a clean, dry gauze pad. This removes any
residual alcohol and any tissue fluid contamination.
 12. Make blood films if requested.
13. Collect blood in the appropriate collection tubes and mix as needed. If an insufficient
specimen has been obtained because the blood flow has stopped, repeat the puncture
at a different site with all new equipment. CLSI recommends the following order of
draw:13 (Box 3-3)
a. Tube for blood gas analysis
b. Slides, unless made from a specimen in the EDTA microcollection tube
c. EDTA microcollection tube
d. Other microcollection tubes with anticoagulants
e. Serum microcollection tubes
14. Apply pressure and elevate the puncture site until bleeding has stopped.
15. Label each specimen with the required information and indicate skin puncture
collection.
Note: Compare the labeled tubes with the identification bracelet for inpatients;
have outpatients verify that the information on the labeled tubes is correct,
whenever possible.
16. Handle the specimens appropriately.
17. Discard all puncture equipment and biohazardous materials appropriately
18. Remove gloves and wash hands.
19. Deliver the properly labeled specimens to the laboratory.

ORDER OF DRAW FOR SKIN PUNCTURE

1. Tube for blood gas analysis
2. Slides, unless made from specimen in the EDTA microcollection tube
3. EDTA microcollection tube
4. Other microcollection tubes with anticoagulants
5. Serum microcollection tubes\

NOTES:

 the best method for blood gas collection in newborns is the INDWELLING UMBILICAL
ARTERY CATHETER
 The site of collection should be warmed to a temperature of no greater than 42° C for no
longer than 2 – 3 minutes (warming can increase the blood flow seven fold)
 Skin should be allowed to air dry after disinfection.
 1.75mm (length of lancet, is preferred to avoid penetrating the bone)
 Depth of incision should be <2.0mm (infants) and 2-3mm (adults). But according to
Rodak’s Hematology; Clinical Principles and Applications 5th edition it should be
(<2mm)
 Distance from the skin surface to the bone/cartilage in the middle finger is (1.5 – 2.4mm)
 First drop of blood should be wiped away (removed residual alcohol and tissue fluid
contamination)
 Capillary Tube length is 7cm (70mm) with a uniform bore of 0.1cm (1mm). It must be
filled three quarters (Rodak’s) or at least 5cm (50mm) (Henry’s Clinical Diagnosis and
Management by Laboratory Methods 23rd Edition)
 Most POC tests require about four to nine drops of capillary blood (approximately 40 –
90uL)

USED IN SEVERAL SITUATIONS:

1. Patients who are severely burned
2. With cancer whose veins are reserved for therapeutic purposes
3. Who are obese and whose veins are too deep to locate
4. Geriatric patients or patients whose veins are inaccessible or very fragile
5. Point of care testing (POCT) in a health care facility
6. Patients performing tests on themselves
7. Special procedures that require capillary blood
  Patients who are severely dehydrated or have poor circulation cannot produce an
adequate capillary puncture blood sample. A patient who is extremely cold will also
not produce adequate blood flow. Patient’s hand needs to be warmed before
capillary puncture if there is any coolness, the best way to warm the hand is with
warm wet washcloth

SAMPLE COLLECTION TRAYS

 Alcohol swabs
 Gauze squares or cotton balls
 Evacuated tube holder
 Evacuated tubes
 Syringe
 Needles
 Butterfly infusion set
 Microcollect equipment
 Tourniquet
 Disposable gloves
 Sharps and/or waste container
 Markin pen
ORDER OF DRAW FOR TUBE COLLECTION
1.) Tube for blood gas analysis
2.) Slides (unless made from sample in the EDTA micro collection tube
3.) EDTA micro collection tube
4.) Other micro collection tubes with anticoagulants
5.) Serum micro collection tubes

III. EVACUATED TUBE SYSTEM

 1949 - Becton Dickinson manufactured the first ever evacuated tube blood collection tube. Under
the brand name VACUTAINER
 1985 - Greiner Bio-One manufactured evacuated tube blood collection tube. Under the brand
name VACUETTE

EQUIPMENT

 Needle
o MULTISSAMPLE NEEDLE - permit several samples to be taken with a single
puncture.
 Adaptor
o Holds the needle in place
 Tubes
o facilitates the drawing of a predetermined volume of blood
o tubes may contain additives designed to stabilize and preserve the specimen prior
to analytical testing.

ORDER OF DRAW FOR EVACUATED TUBE SYSTEM:

  Yellow - - - - - - - - SPS
 Light Blue - - - - - Sodium Citrate
 Red - - - - - - - - - - None
 Green - - - - - - - - Heparin
 Lavender - - - - - - EDTA
 Gray - - - - - - - - - Potassium Oxalate
 Pink - K2 EDTA
 White - EDTA and gel
 Black - Sodium Citrate
 Royal Blue - Sodium Heparin / K2 EDTA
 Tan (Glass) - Sodium Heparin
 Tan (Plastic) - K2 EDTA

ETS COLLECTION TECHNIQUE

 Patient Identification
 If a fasting specimen is required, confirm patient has fasted or eliminated foods from diet
as ordered by physician.
 Position the patient properly. Assemble equipment and supplies.
 Apply a tourniquet and ask the patient to make a fist without vigorous hand pumping.
Select a suitable vein for puncture.
 Cleanse the venipuncture site with 70% isopropyl alcohol. Allow the area to dry
 Anchor the vein firmly.
 Puncture the skin with the needle at approximately a 30- degree angle or less to the arm,
with the bevel of the needle up
 Insert the tube.
 After the tube if properly filled, remove the tube.
 Release the tourniquet
 Remove the needle.
 Patient care

IV. WINGED INFUSION TEST

 used when blood has to be collected from a very small vein
 needles come in 21, 23, and 25 gauge
 needles have plastic wings that aids in insertion of needle

Complications or Problems Encountered in Venipuncture

1. Ecchymosis (Bruise)
 It is caused by leakage of a small amount of blood in the tissue around the puncture site.
Apply direct pressure to the venipuncture site with a gauze pad. Bending the patient’s
arm at the elbow to hold the gauze pad in place is not effective in stopping the bleeding
and may lead to bruising.

2. Hematoma
 results when leakage of a large amount of blood around the puncture site causes the area
to rapidly swell. If swelling begins, the phlebotomist should remove the needle
immediately and apply pressure to the site with a gauze pad for at least 2 minutes.
 Hematomas most commonly occur when the needle goes through the vein or when the
bevel of the needle is only partially in the vein and when the phlebotomist fails to remove
the tourniquet before removing the needle or does not apply enough pressure to the site
after venipuncture. Hematomas can also form after inadvertent puncture of an artery.

3. Fainting (Syncope)
 Before drawing blood, the phlebotomist should always ask the patient whether he or she
has had any prior episodes of fainting during or after blood collection
 If the patient begins to faint, the phlebotomist should remove and discard the needle
immediately, apply pressure to the site with a gauze pad, lower the patient’s head, and
 loosen any constrictive clothing. The phlebotomist should also notify the designated first-
aid providers at the facility. The incident should be documented.
Warning signs: perspiration, beeds on forehead, hyperventilation, loss of color
Vasovagal syncope – falling
4. Diabetic shock
 Experience hypoglycemia because they fasted. If unconscious, let them drink of orange
juice or cola

5. Convulsions
 Patient become unconscious and mild to violent uncontrollable movements

6. Cardiac arrest
 Patient falls unconsciousness, no pulse or respiration, dilated eyes and pale skin

7. Hemoconcentration
 an increased concentration of cells, larger molecules, and analytes in the blood as a result
of a shift in water balance. Hemoconcentration can be caused by leaving the tourniquet
on the patient’s arm for too long.

8. Hemolysis
 The rupture of red blood cells with the consequent escape of hemoglobin—a process
termed hemolysis—can cause the plasma or serum to appear pink or red.
 Can occur if the phlebotomist used too small a needle during a difficult draw; drew the
blood through an existing hematoma; pulled back too quickly on the plunger of a syringe;
forced blood into a tube from a syringe by pushing the plunger; mixed a tube too
vigorously; or contaminated the specimen with alcohol or water at the venipuncture site
or in the tubes.

9. Petechiae
 are small red spots indicating that small amounts of blood have escaped into the skin.
Petechiae indicate a possible hemostasis abnormality and should alert the phlebotomist to
be aware of possible prolonged bleeding.

10. Allergies
 Some patients may be allergic to skin antiseptic substances and adhesive bandages and
tape. The phlebotomist should use hypoallergenic tape or apply pressure manually until
the bleeding has stopped completely. The phlebotomist should also determine if the
patient has a latex sensitivity before the phlebotomy procedure.

11. Nerve Damage

 The phlebotomist must select the appropriate veins for venipuncture and should not
blindly probe the arm with the needle or try to laterally relocate the needle.

12. Seizures
 Patients occasionally experience seizures because of a preexisting condition or as a
response to the needle stick. If a seizure occurs, the phlebotomist should immediately
remove and discard the needle, apply pressure with a gauze pad, and notify the nurse or
designated first-aid providers at the facility.

13. Vomiting
 If the patient begins vomiting, the phlebotomist should provide the patient an appropriate
container and tissues, notify the nurse or designated first-aid providers at the facility, and
ensure the patient’s head is positioned so that he or she does not aspirate vomit.

14. Edema
 Swelling caused by an abnormal accumulation of fluid in the intercellular spaces of the
tissues is termed edema. The most common cause is infiltration of the tissues by the
solution running through an incorrectly positioned intravenous catheter. Edematous sites
should be avoided for venipuncture because the veins are hard to find and the specimens
may become contaminated with tissue fluid.
 15. Nausea
 Make patient as comfy as possible. Instruct to breath slowly, apply cold compress, give
waste basket and have tissue water ready

16. Icteric sample

 Serum/plasma contains large amount of bilirubin
 Patient w/ jaundice

17. Lipemic sample

 Serum/plasma contains large amount of fats and lipids may be due to not fasting
 Milky or white color

SPECIMEN CONSIDERATIONS:
TIME OF SPECIMEN COLLECTION:
1. Fasting
o Non per Orem
o Fasting blood sugar
o 8-12 hours no food intake
2. Post-prandial
o After meal
3. Random
o Any time of time day
4. Basal state
o Early morning after awakening

CLASSIFICATION OF METHODS TO SAMPLE REQUIREMENTS

1. macromethod - 1ml and above
2. Micromethod - 0.1 to 0.9 ml
3. Ultramicromethod - 0.01 to 0.09 mL
4. Nanoliter method - 0.001 to 0.009 mL

FACTORS CONTRIBUTING TO THE VARIATION OF RESULTS

 exercise
 Fasting
 Diet
 Posture or position
 Tourniquet application

1. EXERCISE:
o moderate exercise can cause an increase in:
 blood glucose
 lactic acid
 serum proteins
 muscle enzymes

2. FASTING
 abstinence from eating or drinking for at least 8 hours
 did not undergo fasting blood glucose, potassium and lipids like cholesterol and
neutral fats
 PROLONGED FASTING:
o elevations in serum bilirubin, triglycerides, glycerol, free fatty acids and a
decrease in plasma glucose

3. DIET
• high protein diet = increased urea, ammonia, urates (protein by product)
 • long time vegetarian = decreased LDL, VLDL, total lipid, phospholipid, cholesterol,
TAG
• hyperchylomicronemia = milky serum / plasma
- increases turbidity or lactescence (triglycerides level exceeds 4.6 mml/L
(4.0g/L)

FACTORS CONTRIBUTING TO THE VARIATION OF RESULTS

 Tobacco smoking
 Alcohol ingestion
 Stress
 Drugs
 Age and gender

1. TOBACCO SMOKING
o affects hematological tests such as hemoglobin, hematocrit and blood counts
 affects hematological indexes and oxidative stress by your markers
negatively

2. ALCOHOL INGESTION
• increases plasma concentration of lactate, urate, acetate & acetaldehyde; gamma-
glutamyl transferase (GGT) concentration
• can also affect blood sugar and fat levels, giving inaccurate results to blood tests that
require fasting

3. STRESS / ANXIETY
• affects hormone secretion
• results to hyperventilation leading to a disturbance in acid base balance in the blood

4. DRUGS
 may interfere with liver function test

5. AGE
 bilirubin rises after birth and peaks at about 5 days

6. GENDER
 men have higher reference values compared to women

FACTORS CONTRIBUTING TO THE VARIATION OF RESULTS:

A. LIGHT SENSITIVITY
 some tests are sensitive to light and the component may disintegarte or increase when
exposed to light

B. DIURNAL VARIATION
 some analytes are high in the morning and low at night