Vacutainer and Their Use in Blood Sampling

A vacutainer is a blood collection tube (sterile glass or plastic tube) used to collect blood samples for laboratory testing.

These tubes have a closure that is evacuated to create a vacuum inside the tube thereby enabling a predetermined amount of blood to be withdrawn.

Vacutainer tubes were invented by Joseph Kleiner and Becton Dickinson in 1949. Vacutainer® is a registered trademark of Becton, Dickinson, and Company which manufactures and sells the tubes today.

Vacutainer tubes have a colored rubber stopper at one end which is a kind of code for different types of sampling.

Additives

Vacutainer tubes usually contain some additives to preserve the specimen. As different blood tests measure different substances present within the blood, so the specific test required to be carried out will determine the specific substance that needs to be preserved.

Accordingly, vacutainer tubes contain a variety of anticoagulants which prevent blood from clotting/ coagulation. Some tubes contain coagulants to speed up the clotting of blood. These additives are usually thin film coatings on the inner wall of the tube or in the form of liquid or gel.

The tubes are fitted with a colored plastic or rubber stopper, which indicates the type of additive used in the tube. This color code has a standard universal protocol.

Vacutainer Needles and Action Mechanism

Vacutainer needles are double-ended. The exposed end of the needle is to be inserted into the patient’s vein. The other side of the needle is enclosed in a thin rubber coating. When the needle is inserted into the plastic needle holder, the end of the needle with a rubber coating gets inside the holder, As soon as a vacutainer tube is inserted into the plastic holder, this rubber coating is punctured by the inner needle and the vacuum in the tube draws blood through the needle and into the tube. The vacuum present in the tube determines how much blood will enter the tube. Once that level is obtained, the blood will automatically stop flowing. The filled tube is then removed and similarly, another tube can be inserted and filled.

It is important that the required amount of blood (level is usually marked over the tube) enters the tube.  Otherwise, the desired proportion of blood to the additive will not be achieved resulting in the fallacious test result. In some cases, the specimen might be rejected by automatic analyzers, if the quantity of blood is less than required.

It is also important to thoroughly mix the blood with the additive by gently inverting the tube several times.

Blood collection tubes get expired because with time the vacuum gets lost and blood will not be sucked into the tube when the needle punctures the cap.

Order of Draw

The term order of draw is the sequence in which tubes should be filled.  The order of draw is important in vacutainer as the needle which pierces the tubes can carry additives from one tube into the next, and cause cross-contamination.

The order of draw is as follows, considering that everything was used in the sample.

1. First – blood culture bottle or tube (yellow or yellow-black top)
2. Second – coagulation tube (light blue top).
3. Third – non-additive tube (red top)
4. Last draw – additive tubes in this order:
   • SST (red-gray or gold top). Contains a gel separator and clot activator.
   • Sodium heparin (dark green top)
   • Lithium heparin (light green top).
   • EDTA (lavender top)
   • Oxalate/fluoride (gray top)

Different Types of Tubes

Purple or lavender top

These tubes have dipotassium/tripotassium salts of EDTA as an anticoagulant. These remove free calcium from the blood by chelation, thus preventing coagulation of blood. They are used when whole blood is needed for analysis such as in complete blood counts (CBC), blood smears, blood typing, Hb electrophoresis, and molecular genetic studies.

Green top

They contain sodium heparin or lithium heparin. Heparin acts as an antithrombin thus inhibiting the coagulation of blood. They are used when plasma is to be used rather than serum. They are used for peripheral blood or bone marrow for cytogenetic studies.

Gray top

These tubes have sodium fluoride and potassium oxalate.  Oxalate is an anticoagulant. Fluoride inhibits red cell glycolytic enzymes. As a result, glucose is not used up during storage. Hence it is useful for blood sugar assay.

Light blue top

3.2% buffered sodium citrate is the anticoagulant used. Sodium citrate binds loosely with free calcium ions to form a calcium citrate complex, thus preventing coagulation of blood. These tubes are used for coagulation assays like prothrombin time (INR) and activated partial thromboplastin time.

Black top

These tubes have 3.8% buffered sodium citrate as the anticoagulant. They are used for Erythrocyte Sedimentation Rate (ESR).

Dark blue top

These tubes contain sodium heparin or EDTA as an additive. They are used for trace metal analysis and toxicology tests.

Light yellow top

These tubes contain sodium polyethanol sulfonate (SPS) and acid-citrate-dextrose (ACD) as an additive. They are used for blood culture specimens, blood bank studies, HLA phenotyping, and parental testing.

Tan top (glass or plastic)

They contain either sodium heparin (glass tubes) or K₂EDTA (plastic tubes). They are lead-free and hence used for lead content determinations.

Red top (glass)

Additive-free vacutainer tubes used for tests for antibodies, chemistry, and drugs.

Vacutainer Tubes with Gel

Some vacutainer tubes have a gel having an intermediate density between blood cells and the serum. During centrifugation, the blood cells sink to the bottom of the tube and are covered by a layer of the gel leaving the serum on top.

When plasma containing blood platelets is required for analysis, a vacutainer that does not contain gel or a clotting agent is used and centrifuged. Tubes containing gel can be easily handled and transported after centrifugation without the blood cells and serum mixing.

• Red top (plastic) – clot activator but no serum separation gel
• Gold or ‘tiger’ red/black top-  They contain clot activator and gel for serum separation
• Orange or gray/ yellow ‘tiger’-  They contain thrombin, a rapid clot activator. They are utilized for STAT serum testing.

Caps

In addition to the color code, the plastic caps also have other indicators.

• Opaque caps indicate vacutainer tubes with a normal vacuum.
• Translucent caps indicate vacutainer tubes with a lower vacuum. These are suitable for elderly patients as the risk of venous collapse during vacuum suction is reduced.

Advantages of Vacutainer Tubes Over Syringes

Vacutainer tubes are widely being used nowadays. They have several advantages over the traditional method of drawing blood using syringes.

• They are safer to use.
• They are easier to use. The tubes come with pre-added additives. The color-coding of the stoppers is universal which makes it easier to understand and communicate amongst the medical fraternity.
• It allows many tubes to be filled at one sitting.
• There is a lower chance of hemolysis.
• Since the stoppers need not be removed for adding blood to the tubes, chances of contamination are reduced.