Specimen Collection and Preparation
Accuracy of laboratory testing depends on the quality of the
specimen submitted. Proper specimen collection, identification,
and transport determine the accuracy and utility of the test
results. Please consult the alphabetical test listings for
information about collection and handling of specimens. If there
are any questions, please call IU Health Bloomington
Laboratory at 812-353-9897 or 812-353-9435 to clarify the
specimen requirements. For a limited number of tests, handling
requirements dictate collection of the specimen only at the
hospital or only during limited hours.
Blood Collection
Plasma and Whole Blood: Draw a sufficient amount of whole
blood into a tube containing the proper anticoagulant.
Immediately invert the tube gently several times to mix. Unless
whole blood is required, separate the plasma from the cells by
centrifugation within 30 minutes. Examples of anticoagulant
collection tubes include the following: green-top (lithium or
sodium heparin), lavender-top (EDTA), light blue-top (sodium
citrate), and lavender-top (K2 EDTA).
Note: If test requires whole blood, do not use a plasma gel
tube. Some drug levels performed on plasma cannot be
performed on plasma from a plasma gel tube.
Instructions for platelet poor plasma:
Some tests require double spinning of the blue top tube to
obtain platelet poor plasma. Centrifuge for 6-10 minutes at
3500 rpm. Transfer plasma, leaving a thin layer of plasma
(about 1/8 inch) in the primary tube, into a plastic aliquot
tube. Centrifuge aliquot tube for 6-10 minutes at 3500 rpm.
Transfer plasma into a new aliquot tube again leaving a thin
layer of plasma. Freeze plasma ASAP.
Serum: Draw a sufficient amount of whole blood into a plain,
red-top tube or a gold-top (serum gel) tube. If using a gold-top
(serum gel) tube, gently invert the tube several times to activate
clotting. Allow blood to clot at ambient temperature for 20 to 30
minutes. Centrifuge for 6-10 minutes to separate serum from
clot, depending on the type of centrifuge used. If using a
plain, red-top tube, after centrifugation transfer the serum to a
screw- capped, plastic vial within 1 hour of drawing the
specimen if required. If a specimen is to be centrifuged, do not
stop the centrifuge once started; interrupting the process may
degrade specimen integrity.
Note: There are some tests requiring serum for which gold-top
(serum gel) tubes should not be used. These are
identified in the individual test listings. If you are not
sure if a gold-top (serum gel) tube is acceptable, a plain,
red-top tube is always acceptable.
Blood Specimen Collection Tubes
The following is a list of tubes referred in our specimen
requirements:
• Light Blue-Top (Buffered Sodium Citrate) Tube: This
tube contains 3.2% buffered sodium citrate as an
anticoagulant and is used for coagulation studies.
Note: It is imperative that the tube be completely filled.
The ratio of blood to anticoagulant is critical for
valid results. Immediately after draw, invert tube
8 to 10 times to prevent clotting.
• Red-Top (Plastic) Tube: This tube contains no
anticoagulants and is used for selected chemistry tests
requiring serum and for selected immunohematology
tests requiring clotted blood.
• Gold-Top (Serum Gel [Gel and Clot Activator]) Tube:
This tube contains a clot activator with a gel barrier and
is used for various tests.
Note: After tube has been filled with blood, invert 8 to
10 times to activate clotting; let stand for 20 to 30
minutes. Centrifuge for 6-10 minutes, depending
on the type of centrifuge used. If frozen serum is
required, pour off serum into plastic vial and
freeze. Do not freeze gold tubes.
• Light Green-Top (4.5 mL Lithium Heparin, Plasma Gel)
Tube: This tube contains lithium heparin as an
anticoagulant with a gel barrier for separation and is
used for the collection of heparinized plasma.
Note: After tube has been filled with blood, immediately
invert tube 8 to 10 times to prevent clotting.
Centifuge for 6-10 minutes, depending on the
type of centrifuge used.
• Green-Top (4 mL Lithium Heparin) Tube: This tube
contains lithium heparin as an anticoagulant with no gel
barrier. Plasma from these tubes should be tested or
removed from tube within 4 hours of draw, depending
on the analyte.
• Lavender-Top (EDTA) Tube: This tube contains EDTA
as an anticoagulant and is used for most hematological
tests.
Note: After tube has been filled with blood, immediately
invert tube 8 to 10 times to prevent clotting.
• Lavender-Top (K2 EDTA) Tube: This tube contains
K2EDTA as an anticoagulant and is used for most blood
bank tests.
Note: After tube has been filled with blood, immediately
invert tube 8 to 10 times to prevent clotting.
• Grey-Top (Potassium Oxalate/Sodium Fluoride) Tube:
This tube contains potassium oxalate as an anticoagulant
and sodium fluoride as a preservative. It is used to
preserve glucose in whole blood and for some special
chemistry tests.
Note: After tube has been filled with blood, immediately
invert tube 8 to 10 times to prevent clotting.
• Large Green-Top (10 mL Sodium Heparin) Tube: This
tube contains sodium heparin as an anticoagulant and is
used for the collection of heparinized plasma or whole
blood for special tests.
•
•
•
•
Note: After tube has been filled with blood, immediately
Invert tube 8 to 10 times to prevent clotting.
Royal Blue-Top (K2 EDTA [Lavender Label]) Tube:
This tube contains EDTA anticoagulant and is used for
trace metals analysis.
Note: After tube has been filled with blood, immediately
invert tube 8 to 10 times to prevent clotting.
Royal Blue-Top (Red Label) Tube: This tube contains no
anticoagulant and is used for trace metals analysis.
Note: Refer to the individual metals tests in the
alphabetic test listing to determine the tube type
necessary.
Yellow-Top (ACD) Tube: There are 2 types of ACD
tubes, ACD solution A (8.5 mL) and ACD solution B (6
mL). These tubes contain acid citrate dextrose (ACD)
and are used for special tests.
Note: After tube has been filled with blood, immediately
invert tube 8 to 10 times to prevent clotting.
Special Collection Tubes: Some tests require specific
tubes for proper analysis. Please call IU Health
Bloomington Laboratory Sendouts Department at 812353-5698 prior to venipuncture to obtain the correct
tubes for metals analysis or other tests as identified in the
alphabetic test listings.
Prevention of Backflow During Blood Draw
Since some blood collection tubes contain chemical additives
which could cause adverse patient reactions, it is important to
avoid possible back flow from the tube. To guard against
backflow, observe the following precautions:
• Place patient’s arm in a downward position.
• Hold tube with the stopper uppermost.
• Release tourniquet as soon as blood starts to flow into
tube.
• Make sure tube additives do not touch stopper or end of
the needle during venipuncture.
Recommended Order of Blood Draw
•
•
•
•
Tubes for sterile specimen (blood cultures).
Tubes for coagulation studies (light blue-top).
Red-top serum tube.
Serum tube with or with out additive (gold-top [serum
gel] or trace metal, no additive tube).
• Green-top (lithium or sodium heparin) or plasma gel
tube.
• Lavender-top (EDTA) tube.
• Grey-top (potassium oxalate/sodium fluoride) tube.
Gold-top (serum gel) tubes and VACUTAINER® PLUS serum
tubes contain particulate clot activators and are considered
additive tubes. Therefore, VACUTAINER® PLUS serum tubes
are not to be used as discard tubes before drawing blue-top
citrate tubes for coagulation studies.
Specimen Centrifugation
Centrifuge carriers and inserts should be of the size specific to
the tubes used. Use of carriers too large or too small for the tube
may result in breakage. Ensure that tubes are properly seated in
the centrifuge carrier. Incomplete seating could result in
separation of the Becton Dickinson (BD) Hemogard™ Closure
from the tube or extension of the tube above the carrier. Tubes
extending above the carrier could catch on centrifuge head,
resulting in breakage. Balance tubes to minimize the chance of
breakage. Match tubes to tubes of the same fill level, glass tubes
to glass tubes, plastic tubes to plastic tubes, tubes with BD
Hemogard™ Closure to others with Hemogard™ Closure, gel
tubes to gel tubes, BD VACUTAINER® PLUS tubes with
PLUS tubes, and tube size to tube size.
If the tube requires centrifugation, spin for 6-10 minutes
depending on the type of centrifuge used, at 3,500 rpm unless
otherwise instructed. If a specimen is to be centrifuged, do not
stop the centrifuge once started; interrupting the process may
degrade specimen integrity. Always allow centrifuge to come
to a complete stop before attempting to remove tubes. When
centrifuge head has stopped, open the lid and examine for
possible broken tubes. If broken, use mechanical device such as
forceps or hemostat to remove tubes. Note: Do not remove
broken tubes by hand. See centrifuge
instruction manual for disinfection instructions.
Histology Specimens
Routine Specimens Submitted to Histology for Tissue
Processing:
• Specimen container and requisition must include:
— Patient name
--- Date of birth
— Type of tissue
— Physician name, first and last name
— Body site
— Collection date
• The requisition must also include the physician’s
signature. If a duplicate report is required, please include
the first and last name of the physician who is to receive
the copy.
• Specimens received for routine histological examination
must be submitted in 10% neutral buffered formalin.
Pre-filled containers labeled “10% Formalin” are
provided to all doctors’ offices and nursing units.
Specimens from Surgery are also sent in pre-filled,
labeled formalin containers.
• Transport specimens at ambient temperature.
• Please call the Histology Department at 812-353-5588
with any questions regarding collection procedures.
Renal Stones Submitted to Histology for Tissue Processing
• Send stones to histology with no fixation (do not submit
in formalin).
• Specimen container and requisition must include:
—Patient name
---Date of birth
— Type of tissue
— Body site
— Collection date
— Physician name, first and last name
• The requisition must also include the physician’s
signature. If a duplicate report is required, please include
the first and last name of the physician who is to receive
the copy.
Immunofluorescence Specimens (ie, skin biopsies for vesiculo
bullous disease) Submitted to Histology Requiring Special
Fixatives:
• Please call the Histology Department at 812-353-5588
prior to specimen collection for supplies and further
instructions.
Lymph Nodes Submitted to Histology for Tissue Processing, or
any Specimen Requiring Lymphoma Work-up:
• Send fresh specimen on saline-moistened gauze to the
Histology Department as soon as possible.
• Notify the department immediately.
Cold Knife Cervical Conization Specimens Submitted to
Histology for Tissue Processing:
• Specimens collected onsite and off site may be
submitted fresh (no formalin) to the Histology
Department as soon as possible.
• Cold knife cervical conization specimens may also be
opened by the surgeon, pinned to a paraffin block, and
submitted in formalin.
LEEP Cervical Cone Specimens Submitted To Histology for
Tissue Processing:
• Specimens collected off site should be opened and
placed in a tissue cassette and submitted in formalin.