Why the Order of Draw Prevents Cross-Contamination
The Order of Draw is a standardized sequence for collecting blood specimens into multiple evacuated tubes during a single venipuncture. Adhering to this order is essential to prevent cross-contamination of additives between tubes, which can alter laboratory results and compromise patient care.[1] For phlebotomy certification exams and clinical practice, memorizing and applying the correct order is a high-yield requirement.
Essential Terminology for Additives and Tube Handling
- Additive – A substance (e.g., anticoagulant, preservative, clot activator) pre-filled in a blood collection tube that affects clotting or preserves the specimen.
- Carryover / Cross‑contamination – Transfer of additive residue from one tube to the next, often via the needle or tube stopper, leading to erroneous test results.
- Tube stopper color – Standardized color codes that indicate the additive or the absence of an additive (e.g., light blue = sodium citrate, green = heparin).
- Inversion requirement – Number of gentle inversions needed after collection to ensure adequate mixing of blood with the additive (e.g., 8–10 times for EDTA tubes).[2]
- Blood culture – Specimen collected into special bottles (not a tube) to detect microbial growth; always drawn first to minimize contamination from skin flora.
The CLSI-Standardized Tube Collection Order
The sequence below follows the Clinical and Laboratory Standards Institute (CLSI) GP41 and NCCLS H3-A6 guidelines.[1][3] Tubes are listed from first to last:
- Blood culture bottles (aerobic first, then anaerobic) – no additive; microbial studies.
- Light blue top – sodium citrate (coagulation tests).
- Gold top / Serum separator tube (SST) – clot activator and gel (serum chemistry).
- Red top (plain) – no additive (serum chemistry, serology).
- Dark green top – sodium heparin (plasma chemistry).
- Light green top – lithium heparin with gel (plasma separators).
- Lavender / Purple top – EDTA (hematology, blood bank).
- Pink top – EDTA (blood bank, special testing).
- Gray top – sodium fluoride / potassium oxalate (glucose, lactate).
- Yellow top – ACD solution (immunohematology, special studies).
Note: Specific institutional protocols may vary; always verify the local policy. For exam purposes, the most frequently tested sequence is: Blood culture → Light blue → Gold/Red → Green → Lavender → Gray.[2]
Why This Order Matters
- Blood cultures must be drawn first to reduce the risk of contamination from skin bacteria or from additives in other tubes.
- Light blue (citrate) tubes must follow blood cultures because citrate residue can interfere with coagulation tests if collected after tubes containing EDTA or heparin.
- Serum tubes (gold/red) are drawn before heparin and EDTA tubes because clot activators and gel can contaminate subsequent additives, and heparin can alter serum chemistry results.
- EDTA (lavender) tubes are drawn after heparin because heparin can interfere with EDTA’s anticoagulant action and cause platelet clumping.
- Glycolytic inhibitor tubes (gray) are drawn last because fluoride can disturb enzyme assays and blood gas measurements if carried over.[1]
Patient Safety Risks from Additive Carryover
- Reflux: When using a syringe, failure to follow the order may cause additive to reflux into the vein, leading to patient harm (e.g., citrate-induced hypocalcemia). Always transfer blood from syringe into tubes in the correct order.[4]
- Hemolysis: Vigorous shaking or using the wrong tube order can release intracellular components, invalidating results. Invert gently according to manufacturer instructions.
- Clotting: Inadequate mixing in citrate tubes (light blue) can cause clotting and render coagulation results unusable.
- Needle-in-needle contamination: Using a butterfly needle with a tube holder is acceptable only if the order is followed and a “waste tube” (empty tube without additive) is drawn first when using certain multi-draw needles; waste tubes, if used, are drawn before any additive tube.[3]
Mnemonics and Inversion Requirements for Exams
- Memory aids: Use the mnemonic “Blue Butterfly, Green Grass, Lavender Sky” or “Blood Culture, Blue, Gold, Green, Lavender, Gray”.
- Know that light blue (citrate) tubes require a 9:1 blood-to-additive ratio; underfilling or overfilling invalidates coagulation results.
- The pink top (EDTA for blood bank) is drawn after lavendar; it is not typically tested on basic exams, but some certifications include it.
- Do not draw a coagulation tube (light blue) after a heparin tube – heparin contamination will inhibit clotting factors and produce erroneous results.
- If a patient has only a cholesterol or comprehensive metabolic panel, a single gold top (SST) is sufficient; order of draw is still relevant if multiple tubes are needed.
- On the exam, you may be asked: “Which tube should be drawn third if the first two are blood culture and light blue?” Answer: Gold top (SST) or red top.
- Memorize the inversion requirements for commonly tested tubes: Light blue (3–4 inversions), Gold/Red (5–6), Lavender (8–10), Gray (5–6).[2]
References
- Clinical and Laboratory Standards Institute (CLSI). GP41: Collection of Diagnostic Venous Blood Specimens, 7th ed. (Formerly H3-A6.) Wayne, PA: CLSI; 2017. https://clsi.org/standards/products/general-laboratory/documents/gp41/
- McCall RE, Tankersley CM. Phlebotomy Essentials. 7th ed. Philadelphia: Wolters Kluwer; 2020: Chapter 7 – Blood Collection Equipment, Additives, and Order of Draw. https://books.google.co.ke/books/about/Phlebotomy_Essentials.html?id=3PWrDwAAQBAJ&redir_esc=y
- CLSI (formerly NCCLS). H3-A6: Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard, 6th ed. Wayne, PA: NCCLS; 2007. https://clsi.org/standards/products/hematology/
- Garza D, Becan-McBride K. Phlebotomy Handbook. 10th ed. Boston: Pearson Education; 2017: Chapter 8 – The Art of the Skin Puncture and Venipuncture. https://books.google.je/books?id=Rjav-IpKCwQC&printsec=frontcover#v=onepage&q&f=false