The correct order of draw for venipuncture, set by the Clinical and Laboratory Standards Institute (CLSI), follows this sequence: blood culture bottles first, then light blue (sodium citrate), red or gold (serum), green (heparin), lavender or pink (EDTA), and gray (sodium fluoride) last. This six-step sequence prevents chemical additives in one tube from contaminating the next and throwing off test results.
The Standard Six-Step Sequence
Every tube used in blood collection contains a specific additive (or no additive at all), and those chemicals can interfere with tests run on other tubes. Drawing in the correct order minimizes the chance that trace amounts of one additive carry over into the next tube. Here is the full sequence by tube color:
- Blood culture bottles (yellow/black tops): Always drawn first because they must remain sterile. A typical set includes one aerobic and one anaerobic bottle, each filled with 10 mL of blood for a total of 20 mL per set.
- Light blue top (sodium citrate): Used for coagulation tests like PT and PTT. The tube contains a precise ratio of citrate to blood, so it needs to fill within 10% of its stated draw volume. Underfilling dilutes the blood with too much citrate and skews clotting times.
- Red, gold, or red-speckled tops (serum tubes): Red tops have no additive. Gold tops (SSTs) contain a clot activator and a gel separator. These are drawn before any tubes containing anticoagulants to keep serum chemistry results clean.
- Green tops (heparin tubes): Dark green contains sodium heparin, light or mint green contains lithium heparin. Mint green plasma separator tubes also include a gel barrier. Used for many chemistry panels.
- Lavender, pink, or pearl tops (EDTA tubes): All contain EDTA as an anticoagulant. Lavender and pink are used for complete blood counts and blood bank testing. Pearl tops add a gel separator for molecular testing.
- Gray tops (sodium fluoride/potassium oxalate): Drawn last. The fluoride preserves glucose by stopping red blood cells from consuming it after collection. These are always at the end because oxalate can interfere with many other tests.
Why the Sequence Matters
The order exists to prevent additive carryover, which happens through three main mechanisms. First, blood from one tube can backflow through the needle into the next tube when using a vacuum system. Second, if blood is drawn with a syringe and then distributed into tubes, the syringe needle picks up additive from each tube it contacts and transfers it to the next. Third, directly pouring blood from one tube into another (which should never be done) guarantees contamination.
EDTA contamination is the most studied and most clinically dangerous form of carryover. EDTA tubes use a potassium salt of EDTA, so any trace amount leaking into a serum or chemistry tube artificially raises potassium levels and lowers calcium and magnesium. Research published in Clinical Chemistry and Laboratory Medicine showed that even a tiny carryover of EDTA-containing blood can elevate measured potassium by 35% and reduce calcium by 20%. Larger contamination volumes pushed potassium up by 76% and calcium down by 48%. These aren’t subtle shifts. A potassium result inflated by that much could trigger unnecessary and invasive treatment for a condition the patient doesn’t actually have.
This problem has been documented since 1977, when researchers first reported cases of falsely high potassium and falsely low calcium traced back to EDTA contamination. Decades later, studies measuring EDTA levels in routine samples still find that low-level contamination is surprisingly common and often goes undetected unless the lab specifically tests for it.
The Discard Tube Rule for Butterfly Needles
Winged infusion sets (butterfly needles) have tubing that fills with air before blood reaches the collection tube. If a light blue citrate tube is your first or only tube, that air displaces blood and causes underfilling, which ruins coagulation results. The fix is simple: attach a small discard tube (a 3 mL tube with no additive) first, let it fill just enough to clear the air from the tubing, then switch to the citrate tube. This discard tube is only necessary when the blue top is the first tube in the draw and you’re using a butterfly needle. With a straight needle, no discard tube is needed because there’s no dead space.
Capillary Draws Use a Different Order
If you’re collecting blood from a fingerstick or heelstick rather than a vein, the order reverses. According to WHO guidelines, capillary collection follows this sequence: EDTA (hematology) specimens first, then chemistry specimens, then blood bank specimens. This is essentially the opposite of the venous order, and there’s a specific reason for it. During a skin puncture, platelets activate quickly at the wound site and begin clumping. If you collect the hematology tube last, those clumps can make a complete blood count inaccurate. Drawing the EDTA tube first gets the anticoagulant working on the sample before platelet clumping distorts the results.
Serum Tubes vs. Plasma Separator Tubes
A common point of confusion is the difference between gold-top SSTs and green-top PSTs, since both contain gel separators. The distinction is what sits above the gel after centrifugation. Gold SSTs produce serum (the liquid left after blood clots), while green PSTs produce plasma (the liquid portion with clotting factors still intact, preserved by heparin). In the order of draw, SSTs come before PSTs because serum tubes either have no additive or only a clot activator, while plasma tubes contain heparin, an anticoagulant that could interfere with serum chemistry if carried over.
Remembering the Sequence
Many phlebotomy students use color-based mnemonics to lock in the order. One popular version: “Boys Love Red Green Lights, Go” maps to blood culture, light blue, red/gold, green, lavender, gray. The key principle behind the order is simple: start sterile (blood cultures), move to the most additive-sensitive tube (citrate), draw tubes with no anticoagulant next (serum), then progress through increasingly potent anticoagulants (heparin, EDTA), and finish with the tube whose additive is most likely to cause interference (fluoride/oxalate).
Royal blue top tubes are a special case. They come in two versions: one with no additive and one with EDTA. They’re made with trace-element-free glass for tests like zinc, copper, and lead. The version with no additive is drawn in the serum tube position, and the EDTA version is drawn in the lavender tube position.