PMLS 2-CHAPTER 5 By: SHERWIN P.

GAMMAD,
RMT, LPT, MSPHc
VENIPUNCTURE EQUIPMENT
The first step in learning to perform a venipuncture is knowledge of the needed
equipment.
Considering the many types of blood samples that may be required for laboratory
testing and the risks to both patients and health-care personnel associated with blood
collection, it is understandable that a considerable amount of equipment is required
for the procedure.
Equipment necessary to perform venipunctures includes needles, sharps disposal
containers, holders, evacuated blood collection tubes, syringes, winged blood
collection sets, tourniquets, antiseptic cleansing solutions, gauze pads, bandages, and
gloves. Box 8-1 lists routine venipuncture equipment.
ORGANIZATION OF
EQUIPMENT
An important key to successful blood collection is making sure that all the required
equipment is conveniently present in the collection area.
Maintaining a well-equipped blood collection tray that the phlebotomist carries into the
patient’s room is the ideal way to prevent unnecessary errors during blood collection.
Trays designed to organize and transport collection equipment are available from
several manufacturers (Fig. 8-1).
The phlebotomy tray provides a convenient way for the phlebotomist to carry
equipment to the patients’ rooms.
Except in isolation situations, the tray is carried into the patient’s room. It should be
placed on a solid surface, such as a nightstand, and not on the patient’s bed, where it
could be knocked off.
Only the needed equipment should be brought directly to the patient’s bed.
Mobile phlebotomy workstations with swivel caster wheels have replaced the traditional
phlebotomy tray in some institutions.
With the increased amounts of required equipment necessary for safe phlebotomy, these
versatile mobile workstations can be configured to accommodate phlebotomy trays,
hazardous waste containers, sharps containers, and storage drawers and shelves.
The cart is designed to be wheeled around the hospital and up to the patient’s bedside
to eliminate placing equipment or a phlebotomy tray on the patient’s bed (Fig. 8-2).
In outpatient settings, a more permanent arrangement can be located at the drawing station
(Fig. 8-3).
A blood drawing chair has an attached or adjacently placed stand to hold equipment.
Drawing chairs have an armrest that locks in place in front of the patient to provide arm
support and protect the patient from falling out of the chair if he or she faints.
A reclining chair or bed should be available for special procedures or for patients
who feel faint or ill.
Infant cradle pads or portable infant phlebotomy stations are available for collection of
blood from an infant (Fig. 8-4).
The duties of a phlebotomist include the cleaning, disinfecting, and restocking
of the phlebotomy trays, workstations, and outpatient drawing stations.
Trays should be totally emptied and disinfected on a weekly basis.
Trays also contain equipment for performing the microcollection techniques
EVACUATED TUBE SYSTEM
The evacuated tube system (ETS) (Fig. 8-5) is the most frequently used method for
performing venipuncture.
Blood is collected directly into the evacuated tube, eliminating the need for
transfer of specimens and minimizing the risk of biohazard exposure.
The evacuated tube system consists of a double-pointed needle to puncture the
stopper of the collection tube, a holder to hold the needle and blood collection tube,
and color-coded evacuated tubes.
NEEDLES
Venipuncture needles include multisample needles, hypodermic needles, and
winged blood collection needles.
All needles used in venipuncture are sterile, disposable, and are used only once.
Needle size varies by both length and gauge (diameter).
For routine venipuncture, 1-inch and 1.5-inch lengths are used.
Needle gauge refers to the diameter of the needle bore.
Needles vary from large (16-gauge) needles used to collect units of blood for
transfusion to much smaller (23-gauge) needles used for very small veins.
Notice that the smaller the gauge number the bigger the diameter of the needle.
Needles with gauges smaller than 23 are available, but they can cause hemolysis
when used for drawing blood samples.
They are most frequently used for injections and intravenous (IV) infusions.
Manufacturers package needles individually in sterile twist-apart sealed shields that
are color coded by gauge for easy identification.
Needles must not be used if the seal is broken (Fig. 8-6).
As shown in Figure 8-7, needle structure varies to adapt to the type of collection
equipment being used.
All needles consist of a bevel (angled point), shaft, lumen, and hub.
Needles should be visually examined before use to determine if any structural
defects, such as nonbeveled points or bent shafts, are present.
Defective needles should not be used.
Needles should never be recapped once the shield is removed regardless of whether
they have or have not been used.
Evacuated tube system needles are threaded in the middle and have a beveled point
at each end designed so that one end is for venipuncture and the other end punctures
the rubber stopper of the evacuated blood collection tube.
Evacuated tube system needles are designated as multisample needles.
Multisample needles have the stopper puncturing needle covered by a rubber sheath
that is pushed back when a tube is attached and returns to full needle coverage when
the tube is removed.
This prevents leakage of blood when tubes are being changed.
Various safety shields and blunting devices are available from different
manufacturers.
The BD Vacutainer Eclipse blood collection needle (Becton, Dickinson, Franklin
Lakes, NJ) uses a shield that the phlebotomist locks over the needle tip after
completion of the venipuncture (Fig. 8-8).
Self-blunting needles (PUNCTUR-GUARD, Gaven Medical LLC, Vernon, CT) are
available to provide additional protection against needlestick injuries by making the
needles blunt before removal from the patient.
A hollow, blunt inner needle is contained inside the standard needle.
Before removing the needle from the patient’s vein, an additional push on the final
tube in the holder advances the internal blunt cannula past the sharp tip of the outer
needle.
The blunt cannula is hollow, allowing blood to continue to flow into the tube (Fig. 8-
9).
NEEDLE HOLDERS
Needles used in the evacuated tube collection systems attach to a holder that
holds the collection tube.
Holders are made of rigid plastic and may be designed to act as a safety shield
for the used needle.
Complete units are available that include the holder and a sterile preattached
multisample needle with safety shield. Various types of holders are shown in Figure
8-10.
The Occupational Safety and Health Administration (OSHA) directs that holders
must be discarded with the used needle.
The One-Use Holder by Becton, Dickinson is available with a different threading to
allow a needle to be threaded into the holder only one time (Fig. 8-11).
The entire needle/holder assembly is discarded in a sharps container after use.
Another type of holder that includes the safety device is the Venipuncture Needle-
Pro (Smith Medical, Weston, MA) that consists of a plastic sheath attached by a
hinge to the end of the evacuated tube holder.
The shield hangs free during the venipuncture and is engaged over the needle using a
single-handed technique against a flat surface after the puncture is performed.
The plastic shield can be rotated to provide the phlebotomist a better view of the
venipuncture site and needle placement.
The entire device is discarded in the sharps container (Fig. 8-12A).
Holders that retract the needle include The ProGuard II and the VanishPoint tube
holder.
The ProGuard II safety needle holder (Coviden, Mansfield, MA) uses a one-
handed method to retract the needle into the holder and a cover for the end that is
open to the stopper- puncturing needle.
The VanishPoint tube holder (Retractible Technologies, Little Elm, TX)
automatically retracts the needle by securely closing the end cap while the needle is
still in the patient’s vein (Fig. 8-13A).
The VACUETTE QuickShield Safety Tube Holder comes with a protective cap
and is designed to use with Vacuette multisample needles.
The VACUETTE Quickshield Complete style includes both the holder and a sterile
preattached needle.
After completion of blood collection, the needle is removed from the patient’s vein
and the protective cap is pressed over the needle against a hard surface using a one-
handed technique.
The VACUETTE QuickShield Complete Plus system includes a holder and Vacuette
Visio Plus multisample needle.
In this combination, a “flash” is observed that confirms penetration of the vein.
The BD Vacutainer Passive Shielding Blood Collection Needle (Becton, Dickinson)
and the VACUETTE Premium Safety Needle System (Greiner Bio-One) are the new
generation of safety devices.
The systems include a preassembled multisample needle with safety device and
holder.
In both systems, the insertion of the first tube into the holder releases the safety
shield, which then rests against the patient’s skin.
As the needle is removed from the vein after blood collection, a spring in the holder
causes the safety shield to automatically move forward to cover the needle.
An advantage to these systems is that the needle is immediately covered when
the needle moves out of the vein due to an unexpected move by the patient.
The BD Vacutainer Passive Shielding Blood Collection Needle (Fig. 8-14) has a
safety shield indicator arrow that judges the depth of needle insertion.
The holders have a flat side to lay against the skin for shallow angle of needle entry.
Holders are available to accommodate collection tubes of different sizes.
To provide proper puncturing of the rubber stopper and maximum control, tubes
should fit securely in the holder.
Ribbed pediatric holder inserts can be inserted into the regular size holder for
pediatric tubes.
The rubber-sheathed puncturing end of an evacuated tube system needle screws
securely into the small opening at one end of the holder, and the evacuated blood
collection tube is placed into the large opening at the opposite end of the holder.
The first tube can be partially advanced onto the stopper-puncturing needle in the
holder.
A marking near the top of the holder indicates the distance an evacuated tube may be
advanced into the stopper-puncturing needle without entering the tube and losing the
vacuum (Fig. 8-15).
The tube is fully advanced to the end of the holder when the needle is in the vein.
Blood will flow into the tube once the needle penetrates the stopper.
The flared ends of the needle holder aid the phlebotomist during the changing of
tubes in multiple-sample situations.
Tubes are removed with a slight twist to help disengage them from the needle.
NEEDLE DISPOSAL SYSTEMS
To protect phlebotomists from accidental needlesticks by contaminated needles, a
means of safe disposal must be available whenever phlebotomy is performed.
In recent years, because of the increased concern over exposure to bloodborne
pathogens and mandates by the OSHA, many disposal systems have been developed.
Needles with safety devices activated must always be placed in rigid, puncture-
resistant, leak-proof disposable “sharps” containers labeled BIOHAZARD that
are easily sealed and locked when full.
Syringes with the needles attached, winged blood collection sets, and holders with
needles attached are disposed of directly into puncture-resistant containers (Fig. 8-
16).
Under no circumstances should a needle be recapped.
COLLECTION TUBES
Tubes used for blood collection are called evacuated tubes because they contain a
premeasured amount of vacuum.
The collection tubes used with the evacuated tube system are often referred to as
Vacutainers (Becton, Dickinson, Franklin Lakes, NJ), although they are also
available from other manufacturers.
Use of evacuated tubes with their corresponding needles and holders provides a
means of collecting blood directly into the tube.
Laboratory instrumentation is also available for direct sampling from the evacuated
tubes, providing additional protection for laboratory workers.
The amount of blood collected in an evacuated tube ranges from 1.8 to 15 mL and is
determined by the size of the tube and the amount of vacuum present.
As shown in Figure 8-17, a wide variety of sizes is available to accommodate adult,
pediatric, and geriatric patients.
When selecting the appropriate size tube, the phlebotomist must consider the amount
of blood needed, the age of the patient, and the size and condition of the patient’s
veins.
Using a 23-gauge needle with a large evacuated tube can produce hemolysis,
because red blood cells are damaged when the large amount of vacuum causes them
to be rapidly pulled through the small lumen of the needle.
Therefore, if it is necessary to use a small-gauge needle, the phlebotomist should
collect two small tubes instead of one large tube.
Evacuated tubes are available in glass and plastic.
Tubes are sterile and many are silicone coated to prevent cells from adhering to the
tube, or to prevent the activation of clotting factors in coagulation studies.
Information about the characteristics of a tube is contained on the write-on label
attached to the tube and should be verified by the phlebotomist when special
collection procedures are needed.
Tubes may also contain anticoagulants and additives.
The tubes are labeled with the type of anticoagulant or additive, the draw volume,
and the expiration date.
The manufacturer guarantees the integrity of the anticoagulant and vacuum in the
tube until the expiration date (Fig. 8-18).
As shown in Figure 8-19, evacuated tubes have thick rubber stoppers with a thinner
central area to allow puncture by the needle.
To aid the phlebotomist in identifying the many types of evacuated tubes, the
stoppers are color-coded.
Color coding for routinely used tubes is generally universal; however, it may vary
slightly by manufacturers, and instructions for sample collection usually refer to the
tube color.
This reference to tube color is found on most computer-generated forms.
Each laboratory department has specified sample requirements for the analysis of
particular blood constituents.
Phlebotomists and phlebotomy students should also understand that testing
methodologies and types of instrumentation vary among laboratories.
Therefore, the type of tube collected for a particular test may not be the same in all
facilities.
Direct sampling instrumentation may also be designed to only accept a specific type
of tube, such as a rubber stopper and not a Hemogard closure or vice versa
Two types of color-coded tops are available.
Rubber stoppers may be colored, or a color-coded plastic shield may cover the
stopper, as with the Hemogard Vacutainer System.
Many instruments in the laboratory are designed to identify a blood tube by its
barcoded label and will directly pierce the stopper to aspirate blood into the
instrument for testing.
Hemogard closures provide additional protection against blood splatter by allowing the
stoppers to be easily twisted and pulled off and have a shield over the stopper.
The plastic shield protects the phlebotomist from blood that remains on the stopper after the
tubes are removed from the needle.
The color of the Hemogard closures varies slightly from that of rubber stoppers.
Refer to the front and back inside covers of this book for two manufacturer’s tube guides.
Evacuated tubes fill automatically because a premeasured vacuum is present in the tube.
This causes some tubes to fill almost to the top, whereas other tubes only partially fill.
BD partial-fill tubes are distinguished from regular-fill tubes by translucent colored
Hemogard closures in the same color as regular-fill tubes.
When a tube has lost its vacuum, it cannot fill to the correct level.
Loss of vacuum can be caused by dropping the tube, opening the tube, improper storage,
manufacturer error, using the tube past its expiration date, prematurely advancing the tube
onto the stopper-puncturing needle in the holder, or pulling the needle bevel out of the skin
during venipuncture.
Principles and Use of Color-Coded Tubes
Color coding indicates the type of sample that will be obtained when a particular
tube is used. tests may be run on plasma, serum, or whole blood.
Tests may also require the presence of preservatives, inhibitors, clot activators, or
barrier gels.
To produce these necessary conditions, some tubes contain anticoagulants or
additives, and others do not.
Phlebotomists must be able to relate the color of the collection tubes to the types of
samples needed and to any special techniques, such as tube inversion, that may be
required.
This section discusses the routinely used tubes with regard to anticoagulants,
additives, types of tests for which they are used, and special handling required.
Tests requiring whole blood or plasma are collected in tubes containing an
anticoagulant to prevent clotting of the sample.
Anticoagulants prevent clotting by binding calcium or inhibiting thrombin in the
coagulation cascade (Fig. 8-20).
Ethylenediaminetetraacetic acid (EDTA), citrates, and oxalates are the most
common anticoagulants that work by binding calcium.
Heparin prevents clotting by inhibiting the formation of thrombin necessary to
convert fibrinogen to fibrin in the coagulation process.
All tubes containing an anticoagulant must be gently inverted three to eight times
immediately after collection to mix the contents and to avoid microclot formation.
Before use, tubes with powdered anticoagulant should be gently tapped to loosen the
powder from the tube for better mixing with the blood.
Tubes containing an anticoagulant must be filled to the designated volume draw to
ensure the correc blood-to-anticoagulant ratio and accurate test results.
If a short draw is anticipated, a partial collection tube should be used.
Tubes containing additives also must be gently mixed to ensure effectiveness.
Blood collected in a tube containing an anticoagulant or additive cannot be
transferred into a tube containing a different anticoagulant or additive.
Lavender (Purple) Top
Lavender stopper tubes and Hemogard closures contain the anticoagulant
ethylenediaminetetraacetic acid (EDTA) in the form of liquid tripotassium or spray-
coated dipotassium ethylenediaminetetraacetic acid (K3EDTA or K2EDTA).
Coagulation is prevented by the binding of calcium in the sample to sites on the
large EDTA molecule, thereby preventing the participation of the calcium in the
coagulation cascade (see Fig. 8-20).
Lavender stopper tubes should be gently inverted eight times.
For hematology procedures that require whole blood, such as the complete blood
count (CBC), K2EDTA is the anticoagulant of choice because it maintains cellular
integrity better than other anticoagulants, inhibits platelet clumping, and does not
interfere with routine staining procedures.
K2EDTA tubes are also used for immunohematology testing and blood donor
screening.
The lavender stopper tube should be completely filled to avoid excess EDTA that
may shrink the red blood cells and decrease the hematocrit level, red blood cell
indices, and erythrocyte sedimentation rate (ESR) results.
Lavender stoppe tubes cannot be used for coagulation studies because EDTA
interferes with factor V and the thrombin-fibrinogen reaction.
Pink Top
Pink stopper tubes and Hemogard closure tubes also contain a spray-coated
K2EDTA anticoagulant and are used specifically for blood bank in some facilities.
The tubes are designed with special labels for patient information required by the
American Association of Blood Banks (AABB). Tubes should be inverted eight
times.
White Top
White Hemogard closure tubes containing a spraycoated K2EDTA anticoagulant and
a separation gel are called plasma preparation tubes (PPTs).
This differentiates them from plasma separator tubes that contain heparin as the
anticoagulant.
White Hemogard closure tubes are primarily used for molecular diagnostics but can
be used for myocardial infarction (MI) panels and ammonia levels, depending on the
test methodology and instrumentation.
Tubes should be inverted eight times. Greiner Bio-One EDTA gel tubes have purple
plastic stoppers with yellow tops.
Light Blue Top
Light blue stopper tubes and Hemogard closures contain the anticoagulant sodium
citrate, which also prevents coagulation by binding calcium.
Centrifugation of the anticoagulated light blue stopper tubes provides the plasma used
for coagulation tests.
Sodium citrate (3.2% or 3.8%) is the required anticoagulant for coagulation studies
because it preserves the labile coagulation factors.
Tubes should be inverted three to four times.
The ratio of blood to the liquid sodium citrate is critical and should be 9 to 1 (e.g., 4.5
mL blood and 0.5 mL sodium citrate).
Therefore, light blue stopper tubes must be completely filled to ensure accurate
results.
When collecting coagulation tests on patients with polycythemia or hematocrit
readings greater than 55 percent, the amount of citrate anticoagulant should be
decreased to prevent an increased amount of citrate in the plasma.
The increased citrate in the sample will interfere with the coagulation tests.
The Clinical and Laboratory Standards Institute (CLSI) recommends the use of tubes
containing 3.2 percent sodium citrate to prevent this problem.
If necessary, tubes can also be specially prepared as described in the CLSI guideline.
The glass CTAD tube with a light blue Hemogard closure is designed for specialized
platelet testing of citrated plasma and contains a solution of sodium citrate,
theophylline, adenosine, and dipyridamole.
The purpose of this tube is to minimize in vitro platelet activation and the artificial
entry of platelet factors into the plasma.
Greiner Bio-One CTAD tubes have blue stoppers with yellow tops.
A special blue stopper tube containing thrombin and a soybean trypsin inhibitor is
used when drawing blood and providing serum for determinations of certain fibrin
degradation products.
Black Top
Black stopper tubes containing buffered sodium citrate are used for Westergren
sedimentation rates.
They differ from light blue top tubes in that they provide a ratio of blood to liquid
anticoagulant of 4 to 1.
Specially designed tubes for Westergren sedimentation rates are available.
Green Top
Green stopper tubes and Hemogard closures contain the anticoagulant heparin combined
with sodium, lithium, or ammonium ion.
Heparin prevents clotting by inhibiting thrombin in the coagulation cascade (see Fig. 8-
20).
Green stopper tubes are primarily used for chemistry tests performed on whole blood or
plasma, particularly stat tests or tests that require a fast turn around time (TAT).
Interference by sodium and lithium heparin with their corresponding chemical tests and
by ammonium heparin in blood urea nitrogen (BUN) determinations must be avoided.
In general, lithium heparin has been shown to produce the least interference. Tubes
should be inverted eight times.
Green stopper tubes are not used for hematology because heparin interferes with the
Wright’s stained blood smear.
Heparin causes the stain to have a blue background on the blood smear, making it
difficult to interpret the differential cell identification.
Light Green Top
Light green Hemogard closure tubes and green/ black stopper tubes containing
lithium heparin and a separation gel are called plasma separator tubes (PSTs).
PSTs are used for plasma determinations in chemistry.
They are well suited for potassium determinations because heparin prevents the
release of potassium by platelets during clotting, and the gel prevents contamination
by red blood cell potassium.
Tubes should be inverted eight times.
Greiner Bio- One heparin gel tubes have green plastic stoppers with yellow tops.
Gray Top
Gray stopper tubes and Hemogard closures are available with a variety of
anticoagulants and additives for the primary purpose of preserving glucose.
All gray stopper tubes contain the glucose preservative (antiglycolytic agent)
sodium fluoride. Sodium fluoride maintains glucose stability for 3 days.
Sodium fluoride is not an anticoagulant; therefore, if plasma is needed for analysis,
an anticoagulant must also be present and the tubes must be inverted eight times. In
gray stopper tubes the anticoagulant is potassium oxalate or Na2EDTA, which
prevents clotting by binding calcium.
Gray tubes with only sodium fluoride for serum testing are available.
Sodium fluoride will interfere with some enzyme analyses; therefore, gray stopper
tubes should not be used for other chemistry analyses.
Gray stopper tubes are no used in hematology because oxalate distorts cellular
morphology.
Blood alcohol levels are drawn in gray stopper tubes containing sodium fluoride to
inhibit microbial growth, which could produce alcohol as a metabolic end product.
Tubes with or without potassium oxalate can be used, depending on the need for
plasma or serum in the test procedure.
Royal Blue Top
Royal blue Hemogard closure tubes are used for toxicology, trace metal, and
nutritional analyses.
Because many of the elements analyzed in these studies are significant at very low
levels, the tubes must be chemically clean and the rubber stoppers are specially
formulated to contain the lowest possible levels of metal.
Royal blue stopper tubes are available with a spray-coated silica clot activator for
serum or with K2EDTA or sodium heparin (Greiner Bio-One) for plasma to conform
to a variety of testing requirements.
Tubes with an anticoagulant present must be inverted eight times.
Tan Top
Tan Hemogard closure tubes are available for lead determinations.
They are certified to contain less than 0.1 μg/mL of lead.
The tubes contain the anticoagulant K2EDTA and must be inverted eight times.
Yellow Top
Yellow stopper tubes are available for two different purposes and contain different
additives.
Yellow stopper tubes containing the red blood cell preservative acid citrate dextrose
(ACD) are used for cellular studies in blood bank, human leukocyte antigen (HLA)
phenotyping, and DNA and paternity testing.
The acid citrate prevents clotting by binding calcium and dextrose preserves the red
blood cells.
Sterile yellow stopper tubes containing the anticoagulant sodium polyanethol
sulfonate (SPS) are used to collect samples to be cultured for the presence of
microorganisms. SPS also prevents coagulation by binding calcium (Fig. 8-20).
SPS aids in the recovery of microorganisms by inhibiting the actions of complement,
phagocytes, and certain antibiotics.
Yellow stopper tubes should be inverted eight times.
Light Blue/Black Top
Light blue/black rubber stopper glass tubes contain the anticoagulant sodium citrate
and a polyester gel and a density gradient liquid and are called cell preparation tubes
(CPTs).
CPTs are specialty single tube systems used for whole blood molecular diagnostic
testing so that mononuclear cells can be separated from whole blood and transported
without removing them from the tube.
The mononuclear cells and platelets are separated from the granulocytes and red
bloods cells by the polyester gel and dense gradient liquid when centrifuged.
The tube should be inverted eight times (Fig. 8-21).
Red/Green Top
Red/green rubber stopper glass tubes contain the anticoagulant sodium heparin and a
polyester gel and density gradient liquid and are also CPTs.
This tube is used for whole blood molecular diagnostic testing when the testing
methodology requires heparinized blood.
The tube is mixed by inverting eight times.
Yellow/Gray and Orange Top
Yellow/gray stopper tubes and orange Hemogard closures are found on tubes
containing the clot activator thrombin.
Notice in Figure 8-20 that thrombin is generated near the end of the coagulation
cascade; addition of thrombin to the tube results in faster clot formation, usually
within 5 minutes. Tubes should be inverted eight times.
Tubes containing thrombin are used for stat serum chemistry determinations and on
samples from patients receiving anticoagulant therapy.
Orange Top
Orange stopper tubes containing a thrombin-based medical clotting agent and
separation gel are called rapid serum tubes (RST) (Becton, Dickinson).
RST tubes clot within 5 minutes and are centrifuged for 10 minutes at a high speed
yielding serum in a short period of time, which is ideal for stat serum chemistry
testing.
Tubes should be inverted five times.
Red/Gray and Gold Top
Red/gray stopper tubes and gold Hemogard closures are found on tubes containing a
clot activator and a separation gel.
They are frequently referred to as serum separator tubes (SSTs) (Becton,
Dickinson).
The tubes are spray coated with the clot activator silica to increase platelet
activation, thereby shortening the time required for clot formation.
Tubes should be inverted five times to expose the blood to the clot activator
A polymer barrier gel that undergoes a temporary change in viscosity during
centrifugation is located at the bottom of the tube.
As shown in Figure 8-22, when the tube is centrifuged, the gel forms a barrier
between the cells and serum to prevent contamination of the serum with cellular
materials.
To produce a solid separation barrier, samples must be allowed to clot completely
before centrifuging. Clotting time is approximately 30 minutes.
Samples should be centrifuged as soon as clot formation is complete.
Greiner Bio-One serum gel tubes have red plastic stoppers with yellow tops.
SSTs are used for most chemistry tests and prevent contamination of the serum by
cellular chemicals and products of cellular metabolism.
They are not suitable for use in the blood bank and for certain immunology and
serology tests because the gel may interfere with the immunological reactions.
SSTs are also not recommended for certain therapeutic drug testing.
Red Top
Red stopper plastic tubes and Hemogard closures contain silica as a clot activator.
They are used for serum chemistry tests and serology tests and in blood bank, where
both serum and red blood cells may be used.
The tubes are inverted five times to initiate the clotting process.
Red stopper glass tubes and Hemogard closures are often referred to as clot or plain
tubes because they contain no anticoagulants or additives.
Blood collected in red stopper glass tubes clots by the normal coagulation process in
about 60 minutes.
Centrifugation of the sample then yields serum as the liquid portion.
Red stopper tubes are used for the same purpose as the red plastic tubes.
There is no need to invert glass red stopper tubes.
Red/Light Gray and Clear Top
Red/light gray stopper and clear Hemogard closures are plastic “discard tubes”
because they contain no anticoagulants, additives, or gel.
They are used as a discard tube for coagulation studies, when using a winged blood
collection set, or as a secondary sample collection tube.
No inverting of the tube is required.
Order of Draw
When collecting multiple samples and samples for coagulation tests, the order in
which tubes are drawn can affect some test results.
Tubes must be collected in a specific order to prevent invalid test results caused by
bacterial contamination, tissue fluid contamination, or carryover of additives or
anticoagulants between tubes.
Transfer of anticoagulants among tubes because of possible contamination of the
stopper-puncturing needle must be avoided (Box 8-2).
This is why tubes containing other anticoagulants or clot activators are drawn after
the light blue stopper tube.
EDTA and heparin can cause falsely increased PT and APTT time results that might
cause a health-care provider to change the dosage of medication or misdiagnose a
patient with a coagulation disorder
Tubes containing EDTA, which can bind calcium and iron, should not be drawn
before a tube for chemistry tests on these substances.
Contamination of a green, gold, or red stopper tube designated for sodium,
potassium, and calcium determinations with EDTA, sodium citrate, or potassium
oxalate would falsely decrease the calcium an elevate the sodium or potassium
results.
Tubes containing heparin should not be drawn before a tube for serum specimens.
Holding blood collection tubes in a downward position so that the tube fills from the
bottom up helps avoid the transfer of anticoagulants from tube to tube.
When sterile samples, such as blood cultures, are to be collected, they must be
considered in the order of draw.
Such samples are always drawn first in a sterile bottle or tube to prevent microbial
contamination of the stopper puncturing needle from the unsterile stoppers of tubes
used for the collection of other tests.
The order of draw as recommended by the CLSI for both the evacuated tube system
and when filling tubes from a syringe is:
● Blood cultures (yellow stopper tubes, culture bottles)
● Light blue stopper tubes (sodium citrate)
● Red/gray, gold stopper tubes (serum separator tubes), red stopper plastic tubes
(clot activator), and red stopper glass tubes
● Green stopper tubes and light green (plasma separator tubes) (heparin)
● Lavender stopper tubes (EDTA)
● Gray stopper tubes (potassium oxalate/sodium fluoride)
● Yellow/gray or orange stopper tubes (thrombin clot activator)
Other colored stopper tubes that contain EDTA such as the pink, white, royal blue,
and tan should be drawn in the same order as the lavender stopper tube.
If the royal blue stopper tube contains the anticoagulant sodium heparin, it should
be drawn in the same order as the green stopper tubes.
When the royal blue stopper tube does not contain an anticoagulant, it should be
drawn in the same order as serum tubes.
SYRINGES
Syringes are often preferred over an evacuated tube system when drawing blood
from patients with small or fragile veins.
The advantage of this system is that the phlebotomist is able to control the suction
pressure on the vein by slowly withdrawing the syringe plunger.
Syringes consist of a barrel graduated in milliliters (mL) or cubic centimeters (cc)
and a plunger that fits tightly within the barrel, creating a vacuum when retracted
(Fig. 8-23).
Syringes routinely used for venipuncture range from 2 to 20 mL, and a size
corresponding to the amount of blood needed should be used.
Needles used with syringes are attached to a plastic hub designed to fit onto the
barrel of the syringe.
They are also individually packaged, sterile, and color coded as to gauge size.
Routinely used syringe needles range from 21 to 23 gauge with 1-inch and 1.5-inch
lengths.
Needle protection devices are available for hypodermic syringe needles similar to
evacuated tube needles.
The Hypodermic Needle-Pro (Smiths Medical, Westin, MA) is a syringe needle with
a disposable plastic sheath attached by a hinge (Fig. 8-24).
The sheath hangs free during the venipuncture and is engaged over the needle by
pressing the sheath against a flat surface after the procedure is complete.
The Safety Glide hypodermic needle by Becton, Dickinson (Fig. 8-25) has a
movable shield that the phlebotomist pushes along the cannula with the thumb to en
- close the needle tip after the venipuncture.
Becton, Dickinson also has an Eclipse hypodermic needle that employs a shield that
the phlebotomist locks over the needle tip after completion of the venipuncture
procedure (Fig. 8-26).
The entire needle and syringe assembly is discarded in the designated sharps
container.
Blood drawn in a syringe is immediately transferred to appropriate evacuated tubes
to prevent the formation of clots.
It is not acceptable to puncture the rubber stopper with the syringe needle and allow
the blood to be drawn into the tube.
A blood transfer device provides a safe means for blood transfer without using the
syringe needle or removing the tube stopper (Fig. 8-27).
It is an evacuated tube holder with a rubber-sheathed needle inside.
After blood collection, the syringe tip is inserted into the hub of the device and
evacuated tubes are filled by pushing them onto the rubber-sheathed needle in the
holder as in an evacuated tube system as shown in Figure 8-28.
The entire syringe/holder assembly is discarded in the sharps container after use.
Only syringes with built-in needle protection shields should be used with this
system.
The shield must be activated immediately when the needle is removed from the vein
to avoid accidental needle sticks.
When tubes are filled from a syringe, CLSI recommends the tubes be filled in the
same order as recommended for the order of draw for the evacuated tube system.
However, the personnel in some institutions believe that, because the portion of
blood possibly contaminated by tissue thromboplastin is the first portion to enter the
syringe, it is the last to be expelled.
To avoid microclot formation in anticoagulated tubes, the order for tube fill from a
syringe in these institutions would be:
● Sterile samples (blood cultures)
● Light blue stopper tubes
● Lavender stopper tubes
● Green stopper tubes
● Gray stopper tubes
● Red, SST, or orange stopper tubes
At the same time, the possible transfer of anticoagulants and additives among tubes
must also be considered and will be minimized by using the CLSI protocol.
Institutional protocol should be followed.
WINGED BLOOD
COLLECTION
SETS
Winged blood collection sets (or “butterflies” as they are routinely called) are used
for the infusion of IV fluids and for performing venipuncture from very small or
very fragile veins often seen in children and in the geriatric population.
Winged blood collection needles used for phlebotomy are usually 21 or 23 gauge
with lengths of ½ to 3/4 inch. Plastic attachments to the needle that resemble
butterfly wings are used for holding the needle during insertion and to secure the
apparatus during IV therapy.
They also provide the ability to lower the needle insertion angle when working with
very small veins.
To accommodate the dual purpose of venipuncture and infusion, the needle is
attached to flexible plastic tubing that can then be attached to an IV setup, syringe, or
specially designed evacuated tube holders (Fig. 8-29).
The flexible tubing can make the winged blood collection set more difficult to
manage.
The length of the tubing also results in approximately 0.5 mL less blood entering the
first collection tube, which could interfere with coagulation tests.
It is more expensive to use the winged blood collection set than the standard
evacuated tube system, and phlebotomists should avoid becoming overly dependent
on the winged blood collection set for sample collection.
There are several winged blood collection sets with safety devices built into the
system (Fig. 8-30).
The BD Vacutainer Safety-Lok blood collection set (Becton, Dickinson, Franklin
Lakes, NJ) uses a translucent protective shield that covers the needle immediately
after removal from the vein.
After use, the needle is completely retracted into the shield and locked in place by
pushing the shield forward (Fig. 8-30B).
The BD Vacutainer Push Button blood collection set uses in-vein activation of the
needle
The needle is automatically retracted into the device when the phlebotomist pushes
the activation button with the index finger while the needle is still in the vein.
(Fig. 8-30C) The safety device for the Vacuette safety blood collection set (Greiner
Bio-One, Kermsmuster, Austria) is activated by depressing both sides of the stopper
and sliding it until the tip of the needle is retracted and covered by the protective
shield (Fig. 8-30A).
The Puncture Guard winged blood collection (Gaven Medical, Vernon, CT) produces
a safety device that blunts the needle before withdrawal from the vein (Fig. 8-31).
Another needle set is the Monoject Angel Wing safety needle (Kendall, Mansfield,
MA). When the needle is withdrawn from the vein, a stainless steel safety shield is
activated and locks in place to cover the needle.
COMBINATION SYSTEMS
The S-Monovette Blood Collection System (Sarstedt, Inc., Newton, NC) is an
enclosed multisampling blood collection system that includes the blood collection
tube and collection device.
Blood is collected using either an aspiration or vacuum principle of collection with
multisampling needles with preassembled holders, needle protection devices, and a
safety winged blood collection set (Fig. 8-32).
TOURNIQUETS
Tourniquets are used during venipuncture to make it easier to locate patients’ veins.
They do this by impeding venous but not arterial blood flow in the area just below
where the tourniquet is applied.
The distended vein then becomes more visible or palpable.
Tourniquets are available in both adult and pediatric sizes.
The most frequently used tourniquets are flat latex or vinyl strips (Fig. 8-33).
They are inexpensive and may be disposed of between patients or reused if
disinfected.
Flat nonlatex strips are available for patients or phlebotomists allergic to latex.
Tourniquets with Velcro and buckle closures are easier to apply but are more difficult
to decontaminate.
The advantage of the buckle closure is that it stays on the patient’s arm after release
and can be retightened if necessary.
Blood pressure cuffs can be used as tourniquets. They are used primarily for veins
that are difficult to locate.
The cuff should be inflated to a pressure of 40 mm Hg.
This allows blood to flow into but not out of the affected veins.
VEIN LOCATING DEVICES
A variety of portable devices are available to locate veins that are not easily visible,
particularly in the infant, pediatric, and elderly patient where every attempt should
be made to avoid multiple needlesticks.
The Venoscope II (Fig. 8-34) and Neonatal Transilluminator (Venoscope, L. L. C.,
Lafayette, LA) and the Transillumination Vein Locator (VL-U) (Promedic,
McCordsville, IN) use high-intensity LED lights that shine through the patient’s
subcutaneous tissue to highlight the veins by absorbing the light rather than
reflecting it.
The vein stands out as a dark line enabling the phlebotomis to note the direction of
the vein.
The phlebotomist then marks the vein for needle insertion.
The Wee Sight Transilluminator (Philips Children’s Medical Ventures, Monroeville,
PA) is designed f or a baby’s tiny veins.
It does not emit heat, making it safe for an infant’s delicate skin.
The Vena-Vue (Biosynergy, Elk Grove Village, IL) uses liquid crystal thermography
to locate veins.
The device is placed on the skin like a bandage to cool the skin.
Veins, because they emit heat, will become visible to the phlebotomist.
The Vein Entry Indicator
Device (VEID) (Vascular Technologies, Ness-Ziona, Israel) uses a sensor technology
to indicate correct insertion of a catheter needle in a vein.
The device emits a continuous beeping signal indicating a change of pressure when
the needle penetrates a vein.
The beeping signal stops when the needle exits the vein.
GLOVES
OSHA mandates that gloves must be worn when collecting blood and must be
changed after each patient.
Under routine circumstances, gloves do not need to be sterile.
To provide maximal manual dexterity, they should fit securely.
Gloves are available in several varieties, including powdered and powder-free, and
latex and nonlatex (vinyl, nitrile, neoprene, and polyethylene) (Fig. 8-35).
Gloves with powder are not recommended because the powder can contaminate
patient samples and cause falsely elevated calcium values.
The glove powder can also cause sensitization to latex.
Allergenic latex proteins are absorbed on the glove powder, which become airborne
and can be inhaled when the gloves are put on and taken off.
Cotton glove liners can be worn under latex gloves for persons that develop an
allergic dermatitis to gloves.
Hands must be washed after removing gloves to prevent the transmission of
bloodborne pathogens and to decrease the time of latex exposure and transmission of
latex proteins to other parts of the body.
PUNCTURE SITE
PROTECTION
SUPPLIES
The primary antiseptic used for cleansing the skin in routine phlebotomy is 70
percent isopropyl alcohol.
This is a bacteriostatic antiseptic used to prevent contamination by normal skin
bacteria during the short period required to perform collection of the Sample For
collections that require additional sterility, such as blood cultures and arterial
punctures, the stronger antiseptics such as iodine or chlorhexidine gluconate (for
patients allergic to iodine) are used to cleanse the area (Fig. 8-36).
To preven skin discomfort, iodine should always be removed from the patient’s skin
with alcohol after a phlebotomy procedure.
2 2-inch gauze pads are used for applying pressure to the puncture site after the
needle has been removed.
Gauze pads can also serve as additional protection when folded in quarters and
placed under a bandage.
A bandage or adhesive tape is placed over the puncture site when the bleeding has
stopped. Latex-free tape should be used for persons who are allergic to adhesive
bandages.
Self-adhering gauze bandages (Coban) are available for elderly patients, for use after
arterial punctures or for patients with prolonged bleeding after venipuncture.
It is not recommended to use cotton balls to apply pressure because the cotton ball
fibers can stick to the venipuncture site and may cause bleeding to begin again when
the cotton is removed.
Patients should be instructed to remove the bandage in about an hour (Fig. 8-37).
ADDITIONAL SUPPLIES
Clean glass slides may be needed to prepare blood films for certain hematology tests.
Biohazard bags should be available for transport of samples based on institutional
protocol.
Alcohol-based hand sanitizers are an acceptable substitute for hand washing when
the hands are not visibly soiled.
Wall-mounted hand sanitizers are available in all health-care settings with either gels
or foams.
Carrying personal bottles of hand sanitizers provides a convenient method of
decontamination that is readily available (Fig. 8-38).
The final piece of equipment needed by the phlebotomist is a pen for labeling tubes,
initialling computer-generated labels, or noting unusual circumstances on the
requisition form.
Ensuring the sterility of needles and puncture devices and the stability of evacuated
tubes, anticoagulants, and additives is essential to patient safety and sample quality.
Disposable needles and puncture devices are individually packaged in tightly sealed
sterile containers.
Visual inspection for nonpointed or barbed needles may detect manufacturing
defects.
Manufacturers of evacuated tubes must ensure that tubes, anticoagulants, and
additives meet the standards established by the CLSI.
Evacuated tubes produced at the same time are referred to as a lot and have a
distinguishing lot number printed on the packages.
There is also an expiration date printed on each package.
The expiration date represents the last day the manufacturer guarantees the stability
of the specified amount of vacuum in the tube and the reactivity of the
anticoagulants and additives.
The expiration date should be checked each time a new package of tubes is opened,
and outdated tubes should not be used.
Failure to completely fill tubes (short draws) containing anticoagulants and additives
affects sample quality because the amount of anticoagulant or additive present in the
tube is based on the assumption that the tube will be completely filled.
Possible errors include excessive dilution of the sample by liquid anticoagulants and
distortion of cellular structure by increased chemical concentrations.
Thank You!