The Order of Draw
The sequence blood collection tubes are filled in, and why it matters. The CLSI order, a tube-by-tube color chart, the additive carryover it prevents, and the butterfly discard-tube rule.
When more than one tube is filled from a single venipuncture, the tubes have to be filled in a set sequence. That sequence is the order of draw, and it exists for one reason: to keep the additive in one tube from carrying over into the next and corrupting the result.
The order is set by CLSI, the Clinical and Laboratory Standards Institute, in its venous blood collection standard. The current version is CLSI PRE02, 8th edition, published February 2025, which replaced the document previously known as GP41. The sequence itself did not change with the renumbering.
The correct order of draw
Six positions, from the sterile tube that must come first to the additives that must come last.
Blood culture tube or bottle
Sterile first, so the sample for microbiology is drawn before any tube can introduce contamination.
Coagulation tube (light blue, sodium citrate)
Before any additive tube, so no other additive can carry over and skew clotting results.
Serum tube, with or without clot activator and gel (red, gold / SST)
Clot activator here would interfere with coagulation testing, which is why it follows the citrate tube.
Heparin tube, with or without gel (green, PST)
Heparin would alter both coagulation and EDTA-dependent results, so it comes after serum.
EDTA tube (lavender, pearl, pink)
EDTA strongly binds calcium and carries potassium; drawn late so it cannot contaminate earlier tubes.
Glycolytic inhibitor (gray, sodium fluoride / potassium oxalate)
Last, because oxalate and fluoride interfere with several chemistry and enzyme assays.
Blood collection tubes by color
What each tube contains, how the additive works, and the tests it serves.
| Tube (color) | Additive | How it works | Common tests |
|---|---|---|---|
| Light blue | Sodium citrate (buffered 3.2%) | Reversibly binds calcium to stop clotting; needs a 9:1 blood-to-additive fill | Coagulation: PT/INR, aPTT, D-dimer, fibrinogen |
| Red | None, or clot activator (plastic) | Blood clots; serum is separated by centrifugation | Serum chemistry, serology, infectious-disease serum tests |
| Gold / SST (tiger-top) | Clot activator + gel separator | Gel forms a barrier between serum and cells on spin | Serum chemistry panels |
| Green | Lithium or sodium heparin (+/- gel, PST) | Heparin potentiates antithrombin to inhibit thrombin | Plasma chemistry, STAT electrolytes |
| Lavender / purple | K2 or K3 EDTA | Strongly chelates calcium to prevent clotting | CBC and hematology |
| Pink | K2 EDTA (AABB cross-match label) | Same anticoagulant as lavender, blood-bank labeled | Blood bank: type and screen, crossmatch |
| Gray | Sodium fluoride + potassium oxalate | Fluoride blocks glycolysis; oxalate binds calcium | Glucose, lactate |
Blood culture bottles are drawn before any of these tubes. Exact stopper shades and additive formulations vary by manufacturer; check the tube label and your laboratory's own instructions.
Why the sequence exists
As tubes are changed on the needle, a trace of additive can cling to the needle and transfer into the next tube. Put the wrong tube second and that carryover contaminates the sample. The classic failure is EDTA, the anticoagulant in the lavender tube: carried into a chemistry tube it binds calcium and adds potassium, so calcium reads falsely low and potassium falsely high. Clot activator from a serum tube, carried into a coagulation tube, interferes with the clotting result.
The sequence is arranged so each additive sits downstream of the tests it would corrupt. The sterile blood culture comes first so nothing can introduce contamination before it. The citrate coagulation tube comes before any other additive. The additive tubes that cause the most trouble, EDTA and fluoride/oxalate, come last.
The discard tube rule for winged sets
Winged (butterfly) collection sets have a length of tubing that holds air. If a light blue coagulation tube is the first tube drawn, that air takes up part of the tube's vacuum, so the citrate tube under-fills and the 9:1 blood-to-anticoagulant ratio breaks. An under-filled citrate tube can produce falsely prolonged coagulation results.
The fix is a discard tube: draw one tube first to flush the air out of the tubing dead space, then draw the coagulation tube. The discard tube does not need to fill completely, only enough to clear the tubing. It is only needed when the coagulation tube would otherwise be first.
Does the order of draw still matter?
Follow it every time. It is the mandated standard of practice and it costs nothing. The evidence behind it, though, is more nuanced than the usual telling.
With modern closed evacuated tube systems, where blood never leaves a sealed path, controlled studies have struggled to reproduce additive carryover at all. One 2021 study found that reversing the sequence did not cause detectable EDTA contamination even in routine workflow, and a systematic review the same year concluded the effect of order of draw is negligible with closed systems. The documented, clinically dangerous contamination, spurious hyperkalemia and falsely low calcium, is almost entirely an open-system phenomenon: blood dispensed from a syringe or winged set into open tubes.
So the order of draw is cheap insurance and correct practice, and potassium is the analyte most at risk when carryover does happen. But in a properly used closed system, technique at the point of collection matters more than the sequence alone. That is the case for treating collection as a controlled process rather than a checklist.
Collection done as a controlled process
The order of draw is one piece of the pre-analytical phase, where most laboratory errors are made. DS2's mobile phlebotomists collect to the CLSI order of draw and two-identifier patient verification, whether the draw happens in a clinic or a patient's kitchen. The specimen then moves under temperature-controlled courier, in collection kits built with the right tubes for the test, so the sample that reaches the lab is one the lab can trust.
Order of draw questions
What is the order of draw?
The order of draw is the fixed sequence in which blood collection tubes are filled during a single venipuncture. It exists to stop additives in one tube from carrying over into the next and contaminating the sample. The sequence is set by the CLSI venous blood collection standard.
What is the correct order of draw?
Blood culture tube or bottle first, then the light blue sodium citrate coagulation tube, then serum tubes (red or gold/SST, with or without clot activator and gel), then the green heparin tube, then the lavender or pink EDTA tube, and finally the gray sodium fluoride/potassium oxalate tube.
Why does the order of draw matter?
Small amounts of additive can cling to the needle and transfer from one tube to the next. Drawing tubes out of order lets that carryover contaminate a sample: EDTA carried into a chemistry tube raises potassium and falsely lowers calcium, and clot activator from a serum tube can interfere with coagulation testing. The sequence puts each tube where its additive cannot corrupt the tubes that follow.
What is the CLSI standard for the order of draw?
The current standard is CLSI PRE02, Collection of Diagnostic Venous Blood Specimens, 8th edition (February 2025), which replaced GP41-Ed7 (2017). The recommended order of draw did not change in the renumbering; only the document designation did.
Do you need a discard tube with a butterfly needle?
Yes, when a light blue coagulation tube is the first tube drawn through a winged (butterfly) set. The tubing holds air in its dead space, so a discard tube is drawn first to fill that space with blood. Without it the citrate tube under-fills, the 9:1 blood-to-anticoagulant ratio breaks, and coagulation results can come back falsely prolonged. The discard tube does not need to be completely filled.
Does the order of draw still matter with modern closed tubes?
It remains the mandated standard of practice, and it is cheap insurance worth following every time. The evidence is more nuanced than often stated: controlled studies using closed evacuated systems have struggled to reproduce additive carryover, and a 2021 systematic review called the effect negligible with closed systems. Documented, clinically significant contamination is largely an open-system problem, where blood is dispensed from a syringe into open tubes. Potassium is the analyte most affected when carryover does occur.
References
- 1.CLSI. Collection of Diagnostic Venous Blood Specimens. 8th ed. CLSI standard PRE02. Clinical and Laboratory Standards Institute; 2025 (replaced GP41-Ed7, 2017).
- 2.CLSI. Order of Blood Draw Tubes and Additives: the recommended draw sequence and the additive carryover rationale.
- 3.BD Vacutainer Venous Blood Collection Tube Guide (stopper color, additive, and laboratory use for each tube type).
- 4.Bazzano G, et al. The Order of Draw during Blood Collection: A Systematic Literature Review. Diagnostics (Basel). 2021;11(3):387. (Effect of order of draw is negligible with closed systems.)
- 5.Cornes MP, et al. Order of draw of blood samples does not cause potassium EDTA sample contamination. Biochem Med (Zagreb). 2021;31(1):010902.
- 6.StatPearls (NIH/NCBI Bookshelf). Phlebotomy order of draw: additive carryover mechanism between tubes.
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