The collection of blood samples is foundational to modern diagnostic medicine. Millions of laboratory tests rely on the integrity of collected specimens to provide accurate results that guide patient care and treatment decisions. A strict, standardized protocol called the “Order of Draw” must be followed during collection. This sequence dictates which color-coded blood collection tube is filled first, second, and so on. Following this protocol ensures that chemical components within one tube do not compromise the contents of another, preventing misleading results and misdiagnosis.
Why the Specific Sequence is Required
The necessity for a specific collection sequence stems from the risk of additive carryover, a form of chemical cross-contamination. Most blood collection tubes contain specialized chemicals, such as anticoagulants or clot activators, designed to stabilize the blood for a particular type of test. When the needle is moved from one tube stopper to the next, a small amount of the previous tube’s additive can contaminate the next tube.
If an additive contaminates a subsequent sample, it interferes with the chemical reactions required for accurate testing. For example, an anticoagulant in a tube intended for serum testing prevents the blood from clotting, rendering the sample unsuitable. This protocol is standardized by organizations like the Clinical and Laboratory Standards Institute (CLSI) to mitigate this risk, safeguarding the specimen’s integrity and ensuring accurate laboratory results.
The Official Order of Draw
The established sequence for blood collection is designed to collect the most contamination-sensitive specimens first, progressing to those with the strongest chemical additives last. The first tube collected is always the sterile specimen, typically used for blood cultures (yellow or yellow-black top). Since these tests check for microorganisms, any additive contamination would compromise the results.
Next in the sequence is the light blue-top tube, which contains sodium citrate, an anticoagulant used primarily for coagulation studies, such as Prothrombin Time (PT). Following the light blue tube are the serum tubes, which include those with a red top (no additive) or gold/red-speckled tops (containing a clot activator and gel separator). These tubes are used for chemistry, serology, and immunology tests, and are drawn before strong anticoagulants.
The fourth step involves the tubes containing heparin (green or light green). Heparin works by inhibiting thrombin to prevent clotting, and these tubes are commonly used for STAT and routine chemistry panels. After the heparin tubes, the tubes containing ethylenediaminetetraacetic acid (EDTA) are drawn (lavender or pink). EDTA is a strong anticoagulant that binds to calcium, and because its carryover severely affects most other tests, it is collected late in the process.
Finally, the last tube in the standard order is the gray-top tube, which contains sodium fluoride and potassium oxalate. The sodium fluoride acts as a glycolytic inhibitor, preventing the breakdown of glucose in the sample, which is necessary for accurate blood glucose or blood alcohol testing.
Common Tube Types and Their Uses
The diverse array of blood tests requires different sample preparations, achieved through specific additives in the collection tubes. Anticoagulants prevent blood from clotting so that whole blood or plasma can be analyzed.
Sodium citrate functions by binding to calcium ions, which are necessary for the blood clotting cascade. Because the ratio of blood to citrate is critical for accurate coagulation testing, this tube must be filled completely.
EDTA is a widely used anticoagulant that works by chelating, or binding, calcium. This additive is primarily used for hematology tests, such as a complete blood count, because it preserves the cellular components of the blood.
Conversely, serum separator tubes (SSTs) contain clot activators, often silica particles, which accelerate the natural clotting process. This allows the blood to separate into a clotted mass and a clear fluid called serum, used for many routine chemistry tests. The gel separator within the SST then acts as a barrier, physically separating the serum from the blood cells after centrifugation.
Heparin is an anticoagulant that works by inhibiting thrombin, an enzyme that converts fibrinogen into fibrin during clotting. These samples yield plasma and are frequently used for chemistry tests where a quick turnaround time is desired. Lastly, sodium fluoride stabilizes glucose levels in the sample by stopping the metabolic process of glycolysis, ensuring that the measured glucose concentration remains accurate over time.