Phlebotomy · Routine Blood Collections · 6 min read
Order of Draw, Fill Ratios, Mixing and Hemolysis Prevention
- Identify the correct sequence for the standard venipuncture order of draw established by the Clinical and Laboratory Standards Institute and explain how it prevents additive carryover.
- Describe the mechanical causes of reflux, proper tube inversion protocols, and the critical blood-to-additive fill ratios required to maintain specimen integrity.
- Recognize the procedural causes and diagnostic impacts of hemolysis, and outline the phlebotomist's responsibility when handling compromised specimens.
The Standard Order of Draw and Additive Carryover
Specimen collection requires strict adherence to an established sequence to ensure accurate laboratory results. The Clinical and Laboratory Standards Institute establishes the standard venipuncture order of draw to prevent additive cross-contamination. Additive carryover is the transfer of additive from one collection tube to a subsequent tube via the collection needle. When an additive is inadvertently introduced into an incompatible tube, it alters the chemical composition of the blood sample and compromises testing accuracy.
The standard venipuncture sequence begins with blood culture bottles or yellow-top SPS tubes, which are drawn first. Next, light blue-top sodium citrate tubes are drawn second. Serum tubes, including plain red-top tubes and serum separator tubes, are drawn third in the sequence. Green-top heparin tubes are drawn fourth, followed by lavender-, purple-, or pink-top EDTA tubes as the fifth collection. Finally, gray-top tubes containing sodium fluoride and potassium oxalate are drawn last in the standard order of draw.
Following this exact order protects against specific analytical errors. For instance, carryover of EDTA into serum or heparin tubes causes falsely elevated potassium results. In addition, carryover of EDTA into subsequent tubes causes falsely decreased calcium and magnesium results due to binding. Heparin carryover into a coagulation tube can interfere with testing, particularly by prolonging the activated partial thromboplastin time (aPTT). Furthermore, carryover of silica clot activators into subsequent anticoagulant tubes can induce partial clotting, which ruins specimens intended to remain uncoagulated.
A critical exception involving equipment setup occurs when performing collections with a winged infusion set. When a light blue tube is the first tube collected with a winged infusion set, draw a non-additive or light blue discard tube first to fill the tubing; a discard tube is unnecessary if a preceding tube has already filled the tubing. If a light blue tube is collected first through unfilled butterfly tubing, air in the tubing can cause underfilling unless a discard tube is drawn first. To maintain safety and prevent contamination, a discard tube drawn before a light blue tube must be a non-additive or light blue tube.
Common mistakes
- Failing to draw a non-additive or light blue discard tube when a sodium citrate tube is collected first with a winged infusion set can cause underfilling because of air in the tubing.
- Inverting a collection tube while it is still attached to the multisample needle inside the tube holder, which introduces a severe risk of reflux.
- Shaking an additive tube vigorously rather than performing gentle complete inversions, resulting in mechanical hemolysis.
- Centrifuging a serum tube too quickly before allowing it to clot upright at room temperature for the specified time, which leaves fibrin strands in the separated serum.
Key points
- The Clinical and Laboratory Standards Institute established the venipuncture order of draw (blood cultures/SPS, sodium citrate, serum, heparin, EDTA, sodium fluoride/potassium oxalate) to prevent additive cross-contamination.
- Additive carryover compromises test results; EDTA carryover causes falsely elevated potassium and falsely decreased calcium and magnesium, while heparin prolongs aPTT.
- Prevent reflux into the patient's vein by keeping their arm downward, maintaining collection tubes below the venipuncture site, and releasing the tourniquet once blood flow is established.
- Strict fill ratios must be maintained: light blue tubes require a 9:1 blood-to-anticoagulant ratio (at least 90% full) to avoid falsely prolonged clotting times, while underfilled EDTA tubes cause red blood cell crenation and falsely decreased hematocrit.
- Additive tubes must be gently inverted immediately after removal from the holder (0 times for plain glass red, 3-4 for light blue, 5 for SST/clot activators, and 8-10 for green, EDTA, and gray).
- Hemolysis turns serum or plasma pink, dark red, or brownish, falsely elevating potassium, lactate dehydrogenase, and magnesium; causes include wet alcohol, small needles, excessive syringe pressure, and extreme transport temperatures.
- Phlebotomists do not interpret results; if a specimen is grossly hemolyzed, report the issue to the laboratory or supervisor and follow facility policies for rejection, recollection, and nurse notification.
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