The Clinical and Laboratory Standards Institute (CLSI) defines a specific sequence for filling blood collection tubes during venipuncture, commonly known as the order of draw. This sequence exists to prevent cross-contamination of additives between tubes, which can otherwise produce erroneous results such as falsely elevated potassium or altered coagulation values. The standard order begins with blood culture bottles (yellow SPS) to maintain sterility, followed by the light blue sodium citrate tube for coagulation studies. After the light blue tube come serum tubes (red top or gold/tiger top SST), then the green heparin tube, the lavender EDTA tube, and finally the gray sodium fluoride/potassium oxalate tube. A common mnemonic to remember the sequence is "Stop Light Red Green Yellow Gray," which maps to Sterile, Light blue, Red, Green, Yellow/lavender, and Gray.
Each tube in the order corresponds to a specific additive that determines what tests can be performed on the specimen. Light blue tubes contain sodium citrate, which reversibly binds calcium and is used primarily for coagulation tests such as PT/INR and PTT. These tubes must be filled to a precise 9:1 blood-to-citrate ratio for accurate results. Red top tubes contain no additive and rely on natural clotting, making them appropriate for serum chemistry, serology, and blood bank work. Gold or tiger top SST tubes contain a clot activator and gel separator for serum chemistry testing, while light green PST tubes contain lithium heparin with a gel separator for plasma chemistry, including STAT electrolytes.
The remaining tubes in the order each serve specialized purposes. Green top tubes contain heparin, which potentiates antithrombin III to inactivate clotting factors, and are used for STAT chemistry and plasma tests. Lavender (purple) tubes contain EDTA, a calcium chelator used for hematology testing such as the complete blood count. Gray top tubes contain sodium fluoride and potassium oxalate to inhibit glycolysis, making them appropriate for glucose and lactate testing. EDTA tubes should be inverted eight times after collection to ensure proper mixing. Several specialty tubes also exist, including pink top tubes with K2EDTA for blood bank type and crossmatch, tan top K2EDTA tubes for lead testing, black top buffered citrate tubes for Westergren erythrocyte sedimentation rate, white top PPT tubes with K2EDTA and gel separator for PCR testing, royal blue tubes for trace element and toxicology testing, and yellow top ACD tubes for HLA typing and DNA studies.
Although the order of draw is consistent, two situations require minor modifications. When a winged or butterfly collection set is used and a coagulation tube is drawn first, a small discard tube must be drawn before the light blue citrate tube to clear the air space in the tubing. Otherwise, the under-filled citrate tube will yield inaccurate coagulation results. Additionally, the same CLSI sequence applies when filling tubes from a syringe rather than directly from a tube holder. Additive tubes should always be drawn after non-additive tubes to prevent additive carryover that could contaminate serum tests and alter analyte results.