When drawing blood by the vacutainer method it is important to fill the tubes in the recommended order?

Yes. You do.

Evidence supporting the need for a specific order in which blood collection tubes should be filled was first published over 30 years ago, yet the concept remains elusive to many healthcare professionals with sample collection responsibilities. This article not only reinforces today’s recommended order of draw, but explains how additive carryover during the collection process can alter the test result the laboratory reports. It also discusses what can happen when the order of draw is disregarded. Additive carryover occurs when the needle filling a tube comes in contact with the blood/additive mixture as the tube fills, and transfers a minute amount of blood and additive into the next tube filled. This can occur with both syringe and vacuum draws. In a syringe draw, the carryover occurs with the needle of the safety transfer device. (According to OSHA, blood collected by syringe should be transferred to the tubes using a safety transfer device, not the same needle used to perform the venipuncture.) In a tube holder draw, carryover occurs from the needle within the tube holder as tubes are exchanged. For additive carryover to occur, tubes must be filled in an inverted position so that the blood/additive mixture comes in contact with the needle that pierces the stopper. When patient positioning is such that the tubes are tilted upright relative to a horizontal plane, they fill from bottom to top. When the tube position is inverted relative to horizontal, i.e., the stopper of the tube is lower than the bottom of the tube, the tube fills from top to bottom, contaminating the needle that pierces the stopper. In practice, those who draw blood samples cannot always control the orientation of the tubes as they are filled when using a tube holder. If all tubes could be orientated in such a manner that allowed them to be filled from bottom to top, an order of draw would not be necessary; the interior needle would never come in contact with the blood/additive mixture. However, since patients present a wide range of arm positions, and contamination of the needle that punctures the stopper cannot always be prevented, an order is necessary.

When additives carry over into a different tube type, test results may be dramatically affected. For example:

• Should the EDTA from a lavender-stopper tube, which is rich in potassium, carry over into a tube to be tested for potassium (a green-, red-, gold-, or speckle-top tube), the level of potassium may be falsely elevated leading to life-threatening medical mistakes;
• If a clot activator carries over into a tube to be tested for coagulation studies (blue stopper), the prothrombin time (PT) or activated partial thromboplastin time (aPTT) may be falsely shortened;
• When blood cultures are collected at the same time as other lab work and not filled first, bacteria from the non-sterile stoppers of the tubes can contaminate the bottles used for blood cultures.

Since we know which additives adversely affect which tests, we can arrange the tubes and blood culture bottles so that any carryover is irrelevant. That arrangement is the order of draw. When tubes are filled according to the recommended order of draw, any additive carryover that may occur will have no significant impact on test results. The order is universal for glass and plastic tubes, and irrespective of whether samples are drawn with a tube holder or syringe. The recommended order is as follows:

1. Blood culture tubes
2. Sodium citrate tubes (e.g., blue-stopper)
3. Serum tubes with or without clot activator, with or without gel separator (e.g., red-, gold-, speckled-stopper)
4. Heparin tubes with or without gel (e.g., green-stopper)
5. EDTA tubes (e.g., lavender-stopper)
6. Glycolytic inhibitor tubes (e.g., gray-stopper)

[Note: some facilities have conducted internal studies that support a variation to the recommended order of draw. When an alternative order is supported by reliable evidence, the facility’s protocol should be followed.] This order of draw has changed over the years, the last occurring in 2003. Necessitating the change was the industry-wide transition from glass blood collection tubes to plastic. Whereas glass is a natural clot activator, plastic is not. So in order for blood to clot in safer plastic tubes, manufacturers coat the inside of the tube with a substance to facilitate clotting, like silica particles. However, if the tube following the clot activator tube remained the coag tube, as was the recommended order prior to 2003, carryover threatens the coag results. With the consensus of all major U.S. tube manufacturers, CLSI issued one single change in the order of draw when the venipuncture standard was revised in 2003: serum tubes that used to precede the blue-stopper coag tube were relocated to follow coag tubes. This change was only possible because a myth about tissue thromboplastin was disproved. Prior to 1998, it was thought that the trauma of a venipuncture resulted in the accumulation of tissue thromboplastin in the needle, which, if drawn into a coagulation tube, could alter results. However, because many studies have proven tissue thromboplastin not to affect PT or aPTT results when the citrate tube was the first tube drawn, it was safe for NCCLS (now CLSI) to move the serum tube immediately after the citrate tube in the order of draw. When healthcare professionals with specimen collection responsibilities adhere to the order of draw, patients are more likely to be treated according to results that truly reflect their physiology. Neglecting this key concept can contribute to medical mistakes that can be potentially catastrophic to the patient. Because ignoring the order of draw can have severe consequences to the patient, it is critical that all who draw blood samples adhere to the established order of draw. Do you have to follow the order of draw? Absolutely.

Note: For access to any of the many articles we've written on the order of draw in our newsletter archives, simply enter "Order of Draw" in the search window at the top of this.

Additional note: For an attactive PDF of this article for posting in your facility, visit our Free Stuff page.

What's Your Problem?

AMT has designated Phlebotomists Recognition Week (from Feb. 14-18)

Blood samples must be drawn by phlebotomists in a specific order to avoid cross-contamination of the sample by additives found in different collection tubes. Phlebotomy order of draw is the same for specimens collected by syringe, tube holder, or into tubes preevacuated at the time of collection. The correct order of draw follows:

1. Blood culture tube or bottle
2. Sodium citrate tube (eg, blue closure)
3. Serum tubes, including those with clot activator and gels (eg, red, red-speckled, gold closures)
4. Heparin tube with or without gel (eg, dark green, light green, speckled green closures)
5. EDTA tube with or without gel separator (eg, lavender, pearl, pink closures)
6. Sodium fluoride/potassium oxalate glycolytic inhibitor (eg, gray closure)

The placement of tubes not listed here should take into consideration the potential for their additive to alter results obtained from the next tube if carryover were to occur. Plastic serum tubes containing a clot activator may cause interference in coagulation testing. Only blood culture tubes, glass nonadditive serum tubes, or plastic serum tubes without a clot activator may be collected before the coagulation tube.

Numerous errors can occur during the collection and handling of blood specimens, which pose significant and avoidable risks to the patient and the phlebotomist. When global standards are not fully implemented, it is more likely that patients will be injured during the procedure, biologically representative specimens will not be obtained from patients, and test results will not be comparable from one facility to another.

CLSI’s GP41 —Collection of Diagnostic Venous Blood Specimens provides a descriptive, stepwise process and procedures reflecting the quality system essentials format for diagnostic venous blood specimen collection. Special considerations for collections from vascular access devices, blood culture collection, and collections in isolation environments are included, as well as how to handle emergency situations. An expanded appendix section provides helpful tips for collecting specimens from pediatric and other challenging patients.

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Venipuncture Procedure

ROUTINE VENIPUNCTURE GUIDELINES

MATERIALS

1. Safety Needles, 22g or less

2. Butterfly needles. 21g or less

3. Syringes

4. Vacutainer tube holder

5. Transfer Device 6. Blood Collection Tubes.

• The vacuum tubes are designed to draw a predetermined volume of blood.
• Tubes with different additives are used for collecting blood specimens for specific types of tests.
• The color of cap is used to identify these additives.

7. Tourniquets. Single use, disposable, latex-free tourniquets

8. Antiseptic. Individually packaged 70% isopropyl alcohol wipes.

9. 2×2 Gauze

10. Sharps Disposal Container. An OSHA acceptable, puncture proof container
marked “Biohazardous”.

11. Bandages or tape

SAFETY

1. Observe universal (standard) safety precautions.

2. Observe all applicable isolation procedures.

3. PPE’s will be worn at all times.

4. Wash hands in warm, running water with a appropriate hand washing product,

5. If hands are not visibly contaminated a commercial foaming hand wash product may be used before and after each patient collection.

6. Gloves are to be worn during all phlebotomies, and changed between patient collections.

7. Palpation of phlebotomy site may be performed without gloves providing the skin is not broken.

8. A lab coat or gown must be worn during blood collection procedures.

9. Needles and hubs are single use and are disposed of in an appropriate ‘sharps’ container as one unit.

10. Needles are never recapped, removed, broken, or bent after phlebotomy procedure.

11. Gloves are to be discarded in the appropriate container immediately after the phlebotomy procedure.

12. All other items used for the procedure must be disposed of according to proper biohazardous waste disposal policy.

13. Contaminated surfaces must be cleaned with freshly prepared 10% bleach solution. All surfaces are cleaned daily with bleach.

14. In the case of an accidental needlestick, immediately wash the area with an antibacterial soap, express blood from the wound, and contact your supervisor.

PROCEDURE

1. Identify the patient, two forms of active identification are required.

• Ask the patient to state their name and date of birth.
• This information must match the requisition.

2. Reassure the patient that the minimum amount of blood required for testing will be drawn.

3. Verify that any diet or time restrictions have been met.

4. Order of Draw

• The following order of draw is the approved order as established by CLSI.
• This order of draw should be followed whenever multiple tubes are drawn during a single venipuncture.
• This is to prevent cross contamination by the tube additives that could lead to erroneous results.

1. Blood Culture

2. Light Blue Top (plasma): 3.2% sodium citrate. These tubes are used for coagulation tests and need to be completely filled to ensure the proper ratio of blood to anticoagulant.

3. Red Top (serum): Plain and gel. Used for chemistry and reference tests.

4. Green Top (plasma): With and without gel, contains lithium heparin. These tubes are used primarily for chemistry tests.

5. Lavender or Pink Top (plasma): Contains EDTA. Used primarily for hematology and blood bank testing.

6. Gray Top (plasma): Contains sodium fluoride/potassium oxalate. Used by chemistry for glucose testing.

7. Yellow Top (plasma and cells): Contains ACD solution A or B. Used for Genetics testing.

NOTE: When using a winged blood collection set for venipuncture and a coagulation tube is the first tube needed, first draw a discard tube (plain red top or light blue top). The discard tube does not need to be filled completely.

1. Assemble the necessary equipment appropriate to the patient’s physical characteristics.

2. Wash hands and put on gloves.

3. Position the patient with the arm extended to form a straight-line form shoulder to wrist.

4. Do not attempt a venipuncture more than twice. Notify your supervisor or patient’s physician if unsuccessful.

5. Select the appropriate vein for venipuncture.

• The larger median cubital, basilic and cephalic veins are most frequently used, but other may be necessary and will become more prominent if the patient closes his fist tightly.
• At no time may phlebotomists perform venipuncture on an artery.
• It is not recommended that blood be drawn from the feet .The Providers permission is required to draw from this site.
• Extensive scarring or healed burn areas should be avoided
• Specimens should not be obtained from the arm on the same side as a mastectomy.
• Avoid areas of hematoma.
• If an IV is in place, samples may be obtained below but NEVER above the IV site.
• Do not obtain specimens from an arm having a cannula, fistula, or vascular graft.
• Allow 10-15 minutes after a transfusion is completed before obtaining a blood sample.

6. Apply the tourniquet 3-4 inches above the collection site.

• Never leave the tourniquet on for over 1 minute.
• If a tourniquet is used for preliminary vein selection, release it and reapply after two minutes.

7. Clean the puncture site by making a smooth circular pass over the site with the 70%
alcohol pad, moving in an outward spiral from the zone of penetration.

• Allow the skin to dry before proceeding.
• Do not touch the puncture site after cleaning.

8. Perform the venipuncture

1. Attach the appropriate needle to the hub by removing the plastic cap over the small end of the needle and inserting into the hub, twisting it tight.

2. Remove plastic cap over needle and hold bevel up.

3. Pull the skin tight with your thumb or index finger just below the puncture site.

4. Holding the needle in line with the vein, use a quick, small thrust to penetrate the skin and enter the vein in one smooth motion.

5. Holding the hub securely, insert the first vacutainer tube following proper order of draw into the large end of the hub penetrating the stopper. Blood should flow into the evacuated tube.

6. After blood starts to flow, release the tourniquet and ask the patient to open his or her hand.

7. When blood flow stops, remove the tube by holding the hub securely and pulling the tube off the needle.

8. Gently invert each tube

• Light blue top- invert 3-4 times
• Red and gold tops invert 5 times.
• All other tubes containing an additive should be gently inverted 8-10 times.

9. DO NOT SHAKE OR MIX VIGOROUSLY. If multiple tubes are needed, follow the proper order of draw

9. Place a gauze pad over the puncture site and remove the needle.

10. Activate the safety device and properly dispose of the vacutainer holder with needle attached into a sharps container.

11. Immediately apply slight pressure to the gauxe pad over the venipuncture site..

• Ask the patient to apply pressure for at least 2 minutes.
• When bleeding stops, apply a fresh bandage, gauze or tape.

12. Tubes must be positively identified after filling with a firmly attached patient label.

• The label must include the patient first and last names, DOB,, collection date and time and collectors initials.
• If no patient labels are available, manually label the tubes with the required information. All labels must include two identifiers .
• The tube must be labeled before leaving the patient.

13. Observe special handling requirements

• Some test specimens require special handling for accurate results.
• Refer to the specific test in the online directory for handling and storage requirements.

VENIPUNCTURE PROCEDURE USING A SYRINGE:

1. Place a sheathed needle or butterfly on the syringe.

2. Remove the cap and turn the bevel up.

3. Pull the skin tight with your thumb or index finger just below the puncture site.

4. Holding the needle in line with the vein, use a quick, small thrust to penetrate the skin and vein in one motion.

5. Draw the desired amount of blood by pulling back slowly on the syringe stopper. Release the tourniquet.

6. Place a gauze pad over the puncture site and quickly remove the needle.

7. Immediately apply pressure. Ask the patient to apply pressure to the gauze for at least 2 minutes.

8. When bleeding stops, apply a fresh bandage, gauze or tape.

9. Transfer blood drawn into the appropriate tubes as soon as possible using a Blood Transfer Device, as a delay could cause improper coagulation.

10. Gently invert tubes containing an additive 5-8 times.

11. Dispose of the syringe and needle as a unit into an appropriate sharps container.

INFANT/CHILD PHLEBOTOMY

1. Confirm the patient’s identification

2. Secure patient to Papoose apparatus for stabilization if child is unable to sit upright on their own.

3. Assemble the required supplies

4. Select the collection site and proceed as routine phlebotomy. If the child is old enough, collect blood as in an adult.

TROUBLESHOOTING HINTS FOR BLOOD COLLECTION

If a blood sample is not attainable:

1. Reposition the needle.

2. Ensure that the collection tube is completely pushed onto the back of the needle in the hub.

3. Use another tube as vacuum may have been lost.

4. Loosen the tourniquet.

5. Probing is not recommended. In most cases, another puncture in a site below the first site is advised.

6. A patient should never be stuck more than twice unsuccessfully by a phlebotomist. The Supervisor should be called to assess the patient.