Phlebotomy, the process of drawing blood, is a fundamental healthcare procedure that directly impacts a patient’s diagnosis and treatment. Accurate collection is essential, as errors can lead to misleading laboratory results. The method relies on specialized vacuum tubes designed to preserve the sample for specific testing. Maintaining sample integrity is achieved by following a standardized sequence for filling the collection tubes.
Components of Blood Collection Tubes
Blood collection tubes are sophisticated devices designed to perform specialized functions upon contact with the patient’s blood. The system typically consists of a plastic tube, a pre-measured vacuum that draws the exact volume of blood needed, and a color-coded rubber stopper. The stopper color is the universal indicator of the tube’s chemical additives.
These additives fall into several categories, including anticoagulants, clot activators, and preservatives. Anticoagulants, such as Ethylenediaminetetraacetic acid (EDTA) and Heparin, prevent the blood from clotting, which is necessary for tests that require whole blood or plasma. EDTA works by binding to calcium ions, while Heparin inhibits specific clotting factors like thrombin.
Tubes used for serum analysis contain a clot activator, which speeds up the natural clotting process, sometimes combined with a serum separator gel. The gel forms a stable barrier between the heavier cellular components and the resulting lighter serum after centrifugation. Preservatives, like sodium fluoride, stabilize certain analytes, most notably glucose, by inhibiting cellular metabolism.
The Standardized Order of Draw
The correct sequence for filling blood collection tubes during venipuncture is known as the Order of Draw, a protocol established by organizations like the Clinical and Laboratory Standards Institute (CLSI). This sequence is designed to move from the most sterile collection to tubes containing the most chemically potent additives, minimizing the risk of contamination between samples. Following this order ensures that trace amounts of an additive from one tube do not interfere with the results of a subsequent test.
The sequence begins with the collection of Blood Culture bottles or tubes, used for microbiology testing to detect bacteria. These must be collected first to ensure maximum sterility. Next are the Light Blue-top tubes, which contain sodium citrate, an anticoagulant used for coagulation studies like Prothrombin Time (PT) and Partial Thromboplastin Time (PTT).
Following the coagulation tube are the Serum tubes (Red, Gold, or Speckled top), which may contain a clot activator and/or a separator gel. These tubes are used for chemistry, serology, and immunology testing, where the clotted blood yields serum. The next tube in the order is the Green-top tube, which contains Heparin, an anticoagulant used for plasma chemistry tests.
The sequence then moves to the Lavender or Pink-top tubes, which contain the powerful chelating agent EDTA. These are used primarily for hematology tests like the Complete Blood Count (CBC) and blood bank procedures. Because EDTA is a highly potent anticoagulant that can interfere with many other tests, it is placed late in the order. Finally, the collection ends with the Gray-top tube, which contains sodium fluoride and potassium oxalate, used for glucose and lactate testing due to its ability to prevent glucose breakdown.
Why Additive Contamination Must Be Avoided
Strict adherence to the Order of Draw is necessary because chemical additives are highly concentrated, and even minute carryover can drastically alter test results. Additive carryover occurs when the needle, exposed to the previous tube’s additive mixture, introduces that substance into the next tube. This microscopic contamination has specific, detrimental effects on laboratory analysis.
For example, if EDTA is carried over into a Light Blue-top tube, it will chelate (bind) the calcium required for the coagulation test. This binding action falsely elevates the patient’s clotting times, potentially leading to an incorrect diagnosis of a bleeding disorder. Similarly, the potassium content found in EDTA can artificially increase the measured potassium level in a subsequent serum chemistry tube, a common error known as pseudohyperkalemia.
Heparin, an anticoagulant, can interfere with coagulation assays if carried over. Clot activators can also partially activate clotting in an anticoagulant tube, leading to microclots that compromise the sample. Furthermore, the glycolytic inhibitor in the Gray-top tube, sodium fluoride, can inhibit enzyme reactions in certain chemistry tests if introduced into a previous tube. The sequence ensures that the most interfering additives are collected last to maintain the accuracy of all preceding samples.
Variations in Collection Technique
While the vacuum tube system is the most common method, variations in the collection technique necessitate slight adjustments to the standard Order of Draw to preserve sample quality.
Syringe Collection
One common variation involves drawing blood using a syringe, which is then transferred into the collection tubes using a safety transfer device. In this scenario, the blood is collected all at once, but the tubes must still be filled from the syringe in the exact same standard order to prevent additive carryover from the transfer device needle between tubes.
Capillary Collection
Another procedural variation is the collection of capillary blood, often referred to as a fingerstick or heel stick, which yields a much smaller volume of blood. The order for capillary collection is different because blood begins to clot much faster outside of a vein, and the risk of platelet clumping is higher. For capillary draws, the tubes containing EDTA are collected first to ensure the hematology sample is obtained before micro-clotting can occur.
The capillary order often follows EDTA (Lavender-top), then other additive tubes (like Heparin), and finally, serum tubes. Regardless of the collection method, a required step after filling is the gentle inversion of each tube a specific number of times, typically between three and ten, depending on the additive. This necessary action ensures that the blood thoroughly mixes with the additive to prevent either clotting or the formation of an inhomogeneous sample, which would also lead to unreliable laboratory results.