BLOOD SPECIMEN COLLECTION

CONTAINERS
Red top tube​: Contains no anticoagulant or preservative.
Use: Serum or clotted whole blood, Serum must be separated from cells within 45 minutes of venipuncture. Send
serum in plastic transfer tube.

Gold top (serum separator, ​SST) ​tube​- Contains clot activator and gel for separating serum from cells, but not
anticoagulant. Do not use serum separator tubes to submit specimens for which tricyclic antidepressant levels are
requested.
Use: Serum. May be used for assays requiring serum unless otherwise stated. Separate serum from cells within
45 minutes of venipuncture, Serum may be sent in the tube with an intact barrier or in a plastic transfer tube.

Lavender top tube​: Contains liquid K3 EDTA.

Use: EDTA whole blood or plasma. Send plasma in plastic transfer tube labeled "plasma, EDTA." Send
whole blood in lavender top tube.

Gray top tube​: Contains sodium fluoride, a preservative, and (blood/serum) potassium oxalate, an
anticoagulant. Use: Sodium fluoride whole blood or plasma. Send plasma in plastic transfer tube labeled
"plasma, sodium fluoride." Send whole blood in gray top tube.

Blue top tube: ​Contains sodium citrate.

Use: Sodium citrate plasma. Send plasma in plastic transfer tube labeled "plasma, sodium citrate." Send whole
blood in blue top tube.

Dark Green top tube: ​Contains sodium heparin or lithium heparin

Use: Heparinized whole blood or plasma. Send plasma in plastic transfer tube labeled "plasma, sodium heparin" or
"plasma, lithium heparin," Send whole blood in green top tube.

Light (Mint) Green top tube​: Contains Lithium Heparin and gel for separating plasma from cells. Use:
Heparinized plasma. Separate plasma from cells within 45 minutes of venipuncture, plasma may be sent in the
tube with an intact barrier.

Yellow top tube​: Contains 1 mL acid citrate dextrose (ACD) solution.

Use: ACID whole blood. Send whole blood in yellow top tube.

Royal blue top tube​: May contain sodium heparin for trace metal studies.
Use: Heparinized whole blood. Send whole blood in royal blue top tube.

Pink top tube​: Contains K2 EDTA

Use: EDTA whole blood and plasma for Blood Bank testing.

Blood Tube Order of Draw

When collecting multiple samples the following order of draw is recommended:
Blood Cultures
Coagulation tube (blue top)
Chemistry tubes (gold or tiger top SST, plain red)
Heparin Tube (green top)
Hematology tube (lavender top)
Sodium fluoride (gray top)
Note: ​when filling tubes from a syringe, minimize the chance of clotting by changing the order so that the
additive tubes are filled before the non-additive ones.

PROCEDURE

For each test ordered, verify the following information found on the collection label:

1. Correct amount of blood to be drawn (if unsure, contact laboratory personnel). Minimal volume collections can
be achieved by combining specimen collection of multiple tests that can be drawn in the same container.

2. Correct type of tube or container to use (if unsure contact laboratory personnel)
 3. Conditions under which the blood should be drawn and stored during transport to the laboratory (i.e. on ice,
protect from light, etc.)

4. The time at which the specimen should be collected.

PROCEDURE FOR PATIENT IDENTIFICATION:

Identification of the patient is critical. The phlebotomist must ensure that the blood specimen is being drawn from the
individual designated on the request form. The following steps outline the required procedure. Deviation from
procedure is not acceptable.

1. Ask the patient to give his/her full name. Never call the patient by name for identification. If the patient is
unconscious ask the patient’s nurse, relative or family friend to identify the patient by full name if available.

2. Compare this information with the information on the request or test requisition.

3. Using two identifiers, name and date of birth; compare the patient’s name and date of birth on the request with
the patient’s hospital identification bracelet. In the outpatient setting patients do not have armbands, verbally
verify both identifiers with the patient.

4. If all information is correct and consistent, proceed with the venipuncture. Any discrepancies should be
reported to the patient’s nurse or client service representative for correction.

PROCEDURE FOR VENIPUNCTURE:

1. Wash hands with soap and water with friction for 15 seconds or use alcohol based hand rub. Put on fresh
gloves.

2. After appropriate identification of the patient, select a vein site. Note: although the larger and fuller median
cubital and cephalic veins are used most frequently, wrist and hand veins are acceptable for the venipuncture.
Be careful to notice if certain veins or arms are restricted for use as in mastectomy cases. Samples should not be
obtained from lower extremities.

3. Apply the tourniquet and palpate the vein to determine the adequacy of the vein for the amount of blood to be
drawn. Do not draw blood from a site of a hematoma. Palpate the vein away from the hematoma to prevent
erroneous analysis. Loosen the tourniquet while the skin is being cleansed and the appropriate equipment
assembled. All equipment, needles, needle holders and syringes are for single use and should be discarded at the
end of the procedure. Note that prolongation of tourniquet application can produce erroneous test results.

4. Cleanse the skin with a commercially prepared alcohol pad, using a circular motion from the center to the
periphery. When collecting a blood alcohol do not use an alcohol pad. Use a chloroprep one step frepp to
cleanse the site. See blood culture collection procedure for proper cleansing prior to collection of blood
cultures.

5. Allow skin to dry to prevent hemolysis of the blood and to prevent the patient from experiencing a burning
sensation when the venipuncture is performed. If the site must be touched again, it must be cleaned again
before venipuncture.

6. Re-apply the tourniquet three to four inches above the selected site.

7. When using a vacutainer, insert the collection tube into the holder at the stop position.

8. Grasp the patient’s arm firmly using the thumb to pull the skin taunt to anchor the vein. The thumb should be
one to two inches below the venipuncture site.

9. With the bevel up, line the needle with the vein and puncture the vein. Push the tube forward to puncture the
stopper.

10. Remove the tourniquet as soon as the blood flow is established.

11. Fill the tube until the vacuum is exhausted and the blood flow ceases. This ensures correct blood to
anticoagulant ratios and that a proper amount of specimen is available for analysis. Blue stopper tubes
 collected for coagulation studies must always be filled correctly and completely. Lavender stopper tubes
collected for CBC must contain at least one ml of blood. When in doubt, check the STVHS test directory or
contact the department for verification of correct volume.

12. When the draw is from a small vein or a pediatric patient, a “butterfly” may be used.

13. When collecting multiple samples the following order of draw is recommended:
Blood Cultures
Coagulation tube (blue top)
Chemistry tubes (gold or tiger top SST, plain red)
Heparin Tube (green top)
Hematology tube (lavender top)
Sodium fluoride (gray top)
Note: ​when filling tubes from a syringe, minimize the chance of clotting by changing the order so that
the additive tubes are filled before the non-additive ones.

14. Mix the additive tubes immediately after collection by gently inverting the tube 8-10 times. 15.

Instruct the patient to open his/her hand and place a gauze pad lightly over the venipuncture site.

16. Activate the collection safety device. Needles are for single use only. Once removed from the arm, do not
recap the needle, bend it or remove needle from holder or syringe. The needle and holder (syringe) should be
discarded in a sharps container.

17. Apply an adhesive bandage over the site after applying mild pressure to the site. Instruct the patient to leave
the bandage on for 15 minutes and to apply slight pressure for 5 minutes. If the patient is taking an
anticoagulant, hold pressure and assure bleeding has stopped before applying bandage. If any bleeding
problems are noted, call the nurse for assistance.

18. Place the bar coded accession label on the specimen tubes before leaving the patient’s side. Initial the specimen
label. If a bar coded label is not available record the patient name, date of birth, date and time of collection and
phlebotomist identification on each tube drawn.

Notes

All needles are for single use only

Phlebotomists are not to perform arterial sticks. If arterial stick is necessary, call Respiratory for assistance.
A phlebotomist is to attempt a venipuncture no more than twice. If unsuccessful another phlebotomist or
staff member should attempt to obtain the specimen.

Patient identification is critical. If there is any discrepancy between the request and the patient’s armband
information the specimen should not be drawn until the discrepancy is resolved.
Keep your tray within arm’s reach just in case you get into trouble and realize that you need another tube or
a pediatric size tube.
For inpatients, have the patient lie in the bed. Do not place the tray on the patient’s bed. The bedrail may
be lowered to access the patient, but must be returned to the upright position before leaving the patient’s
room.
For outpatients, use a phlebotomy chair with a safety arm.

BLOOD CULTURE COLLECTION

SUPPLIES
• ​Blood culture skin preparation ​kit
• ​BacT-Alert® blood culture ​bottles
- Use one aerobic (light green cap) and one anaerobic (orange cap) bottles for adults for each
request - Use single pediatric (yellow cap) bottle for ​pediatrics
• ​Butterfly needle, blood culture collection device, sterile gauze and alcohol prep. If drawing from a line use
 line draw blood culture ​device.
PROCEDURE
Follow all patient identification procedures as outlined in the routine collection procedures.
1. Place tourniquet on patient's arm and locate the vein to be used.
2. Loosen the tourniquet and proceed with the skin sterilization procedure.
3. Remove Chloraprep FREPP and hold by the center of the handle in a horizontal position with the foam
surface down. Pinch handle to break ampule. Note: Do not continue to squeeze handle.
4. Apply foam surface to area to be cleansed, depress foam against skin surface once or twice to
saturate foam. Cleanse area thoroughly for 30 seconds. Allow to air dry for 30 seconds. Do not
blot or blow on skin to dry. Do not re-palpitate! Blowing on or touching the cleansed site will
contaminate it causing false positive blood cultures that may lead to erroneous patient treatment.
Do not cough or talk over the cleansed site.
5. Proceed with collection of the blood culture specimen using accepted technique. Remove the top from the
blood culture aseptically so as not to contaminate the top of the bottle. Use an alcohol prep to cleanse the top
of the blood culture bottle prior to inoculation with the specimen.

Adults: ​Use aerobic and anaerobic bottle for each set. Draw 10 to 20 ml. For each venipuncture, minimum is 5 ml
and maximum is 10 ml per bottle. Note: If an adult is extremely difficult to collect and only a small amount to blood
is collected, inoculate the aerobic bottle only. Do not use a pediatric bottle for adult collection.

Pediatric patients (infants and under 12 years): ​Draw 1-4 ml of blood and use one pediatric bottle for each
request. Inoculate pediatric bottles with at least 1 ml blood. NICU specimens can be as small as 0.5 ml. Label
the bottle using a computer label or transcribe the full name, date of birth, or medical record number. All bottles
must have initials of the phlebotomist, time of collection, site of collection, and blood culture order of draw
number. Bandage with gauze and pressure tape.

PROCEDURE FOR FINGER PUNCTURE AND BLOOD COLLECTION FROM

INFANTS
A limited number of test procedures can be done on micro blood samples. Micro test procedures generally require
blood sample collection ranging from 0.1-0.6 mL (100-600 uL). In newborns, when the hematocrit may be 60% to
70%, the whole blood sample collected may need to be three times as much as the actual test sample requirement
in order to yield an adequate volume of serum for testing. A minimum of 0.500 mL whole blood is
required in the lavender top ​Microtainer t​ ube for a CBC.
1. Sites for skin puncture
• ​Lateral or medial plantar heel surface (see illustration)
• ​Plantar surface of the big toe
• ​Palmar surface of the last segment of the finger

PROCEDURE FOR INFANT

HEEL PRICK BLOOD SAMPLES

2. Skin puncture precautions

• ​Skin puncture site must not be edematous as accumulated tissue fluid will contaminate the blood specimen. ​• ​Do
not puncture through a previous puncture site. To do so may spread any possible infection at that site. •​ ​Do not
perform skin punctures on the fingers or toes of newborns. The distance from skin surface to bone in
the thickest portion of the last digit of each finger of newborns varies from 1.2-2.2 mm and with
 available lancets the bone could be damaged.
3. Thoroughly cleanse the puncture site area using a sterile alcohol swab. Allow time for alcohol to evaporate or
dry with a sterile gauze pad before the skin is punctured, as residual alcohol will cause rapid hemolysis of the
specimen.
4. Only use a sterile microlancet device to perform skin punctures.
5. Wipe away the first drop of blood (which may contain tissue fluid) with a sterile gauze pad before beginning
the actual blood collection.
6. Collect blood required for the test procedures requested into the appropriate containers. Blood flow will be
enhanced if the puncture site is held downward and gentle or moderate continuous pressure is applied to the
surrounding area. Note: Strong repetitive pressure (i.e., milking) may cause hemolysis, contamination of
specimen with tissue fluid, and clotting of the blood.
7. Specimen collection for Alabama Department of Public Health Neonatal Screening Test Form: •​ ​Collect
specimen directly onto form. Do not collect in capillary tubes and transfer to ​form. •​ ​Touch drop of
blood formed onto the center of the printed circles on the filter paper portion of the
form. Allow blood to saturate the circle so that the white portion inside is no longer visible on either
side of the form. Repeat the procedure for all circles. Avoid a layering technique (i.e., method
whereby the filter paper is touched to several drops of blood). A single large drip of blood should be
used to saturate the filter paper circle. Do not touch or handle the filter paper.
• ​Allow blood filter paper specimen to air dry horizontally for approximately 4 hours at room
temperature.
• ​Deliver specimens to the Specimen Processing Area of the laboratory for mailing to the State
Laboratory.
8. After collection, apply a sterile gauze pad or cotton ball to the puncture site and hold until the bleeding has
stopped. For infants, this process may be accelerated if the foot is elevated.
9. Remove all materials used and dispose of microlancets immediately in needle disposal container provided. 10.
Deliver specimens properly sealed, mixed, and labeled along with appropriately completed requisitions to the
Laboratory Specimen Processing Area.
11. All specimens should be placed in sealed, disposable plastic bags to prevent contamination by breakage
or spillage.

BLOOD PREPARATION PROCEDURES

There are two important guidelines to follow when submitting blood specimens. For some tests. such as chemistry
procedures, fasting samples are often the specimen of choice. Also, because hemolysis and lipemia interfere with
many procedures, please submit samples that are as free from hemolysis and lipemia as possible.

PREPARING SERUM

Serum Preparation From Red Top Tube​. Follow the steps below when preparing a serum specimen for submission.
1. Draw whole blood in an amount 2 1/2 times the required volume of serum so that a sufficient amount of serum
can be obtained. The 5 mL red top tube will yield approximately 2.5 mL serum after clotting and centrifuging.
Label the specimen appropriately.
2. Place the collection tube in the upright position in the rack, and allow the blood to clot at room temperature for
no longer than 30-45 minutes. If clotting fails to occur within 30 minutes, notify the physician. 3. After allowing
clot to form 20-30 minutes, insert the tube in the centrifuge, stopper end up. Operate the centrifuge for 15 minutes
at the speed recommended by the manufacturer. Do not allow prolonged centrifugation as this may cause
hemolysis. When using a bench-top centrifuge, employ a balance tube of the same type containing an equivalent
volume of water. The tube stopper should remain.
4. Turn the centrifuge off and allow it to come to a complete stop. Do not stop it by hand or brake. Remove the
tube carefully without disturbing the contents.
5. Remove the stopper and carefully aspirate all serum from cells, using a separate disposable pipette for each
tube. Place the tip of the pipette against the side of the tube, approximately 1/4 inch above the cell layer. Do
not disturb the cell layer or carry any cells over into the pipette, It cells do enter the pipette, recentrifuge the
entire specimen.
6. Transfer the serum from the pipette into the transfer tube. Inspect the serum for signs of hemolysis and
turbidity by holding it up to the light. Be sure to provide the laboratory with the amount of serum specified. 7.
Label the tube carefully and clearly with all pertinent information or bar code. Unless otherwise indicated, serum
samples may be sent at room temperature. When multiple tests requiring frozen serum are ordered, a plastic
transfer tube should be prepared for each test.
When frozen serum is required, place the plastic transfer tube(s) immediately in the freezer compartment of
the refrigerator. Notify your professional service representative that you have a frozen specimen to be picked
up; A separate frozen sample must be submitted for each test requiring a frozen specimen.

Serum Separator Tubes (SST ). ​Serum separator (Gold, mottled red/gray top) Tubes ​contain clot
activator and gel for separating serum from cells but include no anticoagulant. Adhere to the following steps when
using a serum separator tube; Do not use serum separator tubes to submit specimens for which tricyclic
antidepressant levels, Direct Coombs', Blood Group, and Types are requested.
1. Draw whole blood in an amount 21/2 times the required volume of serum so that a sufficient amount of
serum can be obtained. The 5 mL Gold top tube will yield approximately 2 mL serum after clotting and
centrifuging. The 10 mL mottled red/gray top tube yields approximately 4 mL serum. Label the specimen
appropriately.
2. Gently invert the serum separator tube five times to mix the clot activator and blood.
3. Place the collection tube in the upright position in the rack, and allow the blood to clot at room temperature
for no longer than 30-45 minutes. (Clots usually form in 20-30 minutes.)
4. After allowing the clot to form 20-30 minutes, insert the tube in the centrifuge, stopper end up. Operate the
centrifuge for 15 minutes at the speed recommended by the manufacturer. Do not allow prolonged
centrifugation as this may cause hemolysis. When using a bench-top centrifuge, employ a balance tube of the
same type containing an equivalent volume of water.
5. Turn the centrifuge off and allow it to come to a complete stop. Do not stop it by hand or brake. Remove the
tube carefully without disturbing the contents. Inspect the barrier gel to ensure that it has sealed the serum from
the packed cells. Also, examine the serum for signs of hemolysis (red color) and turbidity (milky or opaque) by
holding it up to the light. Be sure to provide the laboratory with the amount of serum specified. 6. Make sure the
tube is clearly labeled with all pertinent information or bar code.
7. If a frozen specimen is not required, it is not necessary to transfer serum to a plastic transport tube. 8. When
frozen serum is required, always transfer the serum (using a disposable pipette) into a separate, clearly labeled
plastic transfer tube Place the tube immediately in the freezer compartment of the refrigerator, and notify the
professional service representative that you have a frozen specimen to be picked up. Never freeze a glass serum
separator tube. Submit a separate clearly labeled plastic transfer tube for every test requiring a frozen sample.
Unless otherwise indicated, serum samples may be sent at room temperature.

PLASMA PREPARATION
When plasma is required, follow these steps.

1. Always use the proper vacuum tube for tests requiring a special anticoagulant (e.g., EDTA, heparin,
sodium citrate, etc) or preservative.
2. Tap the tube gently to release additive adhering to the tube or stopper diaphragm.
3. Permit the vacuum tube to fill completely. Failure to fill the tube will cause an improper blood-to
anticoagulant ratio and yield questionable test results.
4. To avoid clotting, mix the blood with the anticoagulant or preservative immediately after drawing each
sample. To ensure adequate mixing, slowly invert the tube five to six times using a gentle wrist rotation
motion.
5. Immediately centrifuge the specimen for 5minutes. Do not remove the stopper.
6. Turn the centrifuge off and allow it to come to a complete stop. Do not stop it by hand or brake. Remove the
tube carefully without disturbing the contents.
7. If you do not have a Light Green top tube (Plasma Separator tube), remove the stopper and carefully aspirate
plasma, using a separate disposable Pasteur pipette for each tube. Place the tip of the pipette against the side
of the tube, approximately 1/4 inch above the cell layer. Do not disturb the cell layer or carry any cells over
into the pipette. Do not pour off; use transfer pipette,
8. Transfer the plasma from the pipette into the transfer tube. Be sure to provide the laboratory with the amount of
plasma specified.
9. Label all tubes clearly and carefully with all pertinent information or bar code. All tubes should be labeled
with the patient's full name or identification number as it appears on the test request form or affix bar code.
Also, print on the label the type of plasma submitted (e.g., "Plasma, Sodium Citrate," "Plasma, EDTA," etc).
10. When frozen plasma is required, place plastic transfer tube(s) immediately in the freezer compartment of the
refrigerator, and notify your professional service representative that you have a frozen specimen to be picked
up.