I believe I have mentioned stories like this in previous posts, but there have been many times in the past where I would be at work and see a coworker completely disregard the order of draw. Once, when I was working in the ED, I entered a patient’s room to perform a blood draw. The patient’s nurse was also in the room. I performed my venipuncture and reached for my tubes, and she—trying to be helpful—picked all the tubes up and started handing them to me one at a time.
She handed me the lavender EDTA tube first, and I set it down and asked for the light blue tube. She handed it to me, and I popped it on the hub and let it fill. When it was done, she handed me the green tube. I also set that one down and asked for the red tube. She had been chatting with the patient and not paying close attention, but when I asked for the red tube that time, she looked at the tubes and said something along the lines of “Oh, is this that tube order thing we learned about a while back? I don’t think that really matters.”
Now I know she was a nurse, and not a phlebotomist, but she had to perform blood collections pretty regularly. Anytime one of us phlebotomists were unavailable she would have to do it herself. She said knew about the “order of draw thing,” but when I pulled her aside later to have a conversation about all this, she completely disregarded it.
She told me she learned a little about it in nursing school, and we (the ED staff) also had training on it at the hospital a couple of times a year, but she didn’t think it was important. In that conversation, she said that she drew the lavender tube first because it was always ordered on every patient. The rest she just drew in whatever order she grabbed them in.
Even after my conversation with her about how important the order of draw was, she still brushed it off like it wasn’t a big deal. She said that no one worried about the order of draw except me, and she had never seen anyone have any problems with it. She said that there were lots of things I learned in school that my teachers told me were “super important,” but in real life things are sometimes different.
I was so frustrated, I took it to the department manager, who also seemed to not understand (or care about) the importance of this absolutely critical part of blood collection. She said she would see if they could schedule it in some upcoming training, but I knew it would make no difference. I made what change I could, but when the manager doesn’t care, why should the staff? Even some of the other phlebotomists didn’t care about the order of draw and other important aspects of phlebotomy.
It was overwhelming and difficult as a newer hire in that hospital to see these mistakes and not know how to create change. I knew I was also probably coming off as being annoying bringing the subject up with multiple people. I have had this same experience with several people I have worked with over the years–once even with a senior phlebotomist who was supervising me. Sometimes it was almost word for word the same as what you have just read. With a few of them I managed to explain well enough about how important it is, and they listened, but others didn’t really believe me because “it’s never caused a problem” or “everyone does it this way.” I still have this conversation with people way too often.
What drives me crazy is that phlebotomy is seen as something that everyone can do. That it’s basic. So few people understand the nuances, knowledge and skill that go into it. And what’s more frustrating, is that this nurse (and other people just like her) with many years of experience under her belt had repeatedly learned these things in the past and continued to choose to ignore them, putting people at risk and willingly deviating from the standard of care.
No matter your role, if you’re performing phlebotomy, you need to be knowledgeable about it. The order of draw isn’t optional. Drawing tubes out of sequence causes serious problems. Problems the lab can’t detect.
Why Light Blue Comes Second
Light blue top tubes hold the second position in the order of draw because coagulation testing is extraordinarily sensitive to contamination. After blood cultures, light blue tops come next—before any tube containing other anticoagulants or additives that could interfere with clotting studies.
These tubes are used for coagulation testing—PT/INR, PTT, d-dimer, fibrinogen. Physicians rely on these results for life-or-death decisions: Should we start anticoagulation? Is this patient’s blood too thin? The difference between a therapeutic INR and a dangerous one can be tenths of a point.
If even trace amounts of EDTA (from lavender tops) or heparin (from green tops) get into a light blue tube, they falsely prolong clotting times. The patient’s blood might be normal, but the contaminated specimen makes it look like they have a coagulation disorder.
Light blue tubes come second because contamination is inevitable once you start touching other tubes. The needle picks up trace additives. By placing light blue tubes immediately after blood cultures—before red, gold, green, or lavender tops—we minimize interference with coagulation testing.
The History of Coagulation Testing
In the early 1900s, physicians knew blood clotted but couldn’t measure it reliably. The breakthrough came in 1914 when researchers discovered sodium citrate could prevent clotting by binding calcium—and the effect was reversible. Unlike other substances that destroyed clotting factors, citrate simply locked up calcium temporarily. Add calcium back, and the blood would clot normally.
Dr. Armand Quick developed the prothrombin time (PT) test in 1935, which became the foundation for monitoring anticoagulation therapy. But early tests had a problem: results varied wildly between laboratories. The ratio of blood to anticoagulant had to be precise, or tests were meaningless.
By the 1950s and 1960s, research established that a 9:1 ratio of blood to sodium citrate produced the most accurate results. This was later codified by CLSI (the Clinical and Laboratory Standards Institute) in their H21 guideline for coagulation specimen collection.
What Sodium Citrate Actually Does
Sodium citrate is a salt derived from citric acid—the same acid that gives citrus fruits their tartness. But in blood collection tubes, it serves a very specific purpose: it grabs onto calcium and holds it tight.
Here’s why that matters: blood clotting is a complex chain reaction involving dozens of proteins and enzymes, but calcium is absolutely essential for multiple steps in that cascade. Without calcium, the clotting process can’t proceed. Think of calcium as the key that unlocks several doors in the clotting pathway—without it, those doors stay locked and blood stays liquid.
When blood flows into a light blue tube, the sodium citrate immediately binds to the calcium in the blood through a process called chelation—essentially grabbing onto the calcium ions and holding them tight. It doesn’t destroy the calcium or the clotting factors—it just locks the calcium up so it can’t participate in clotting. The blood stays liquid, which allows it to be tested.
This is different from what happens in other anticoagulant tubes. EDTA (in lavender tops) also chelates calcium, but it binds so tightly that the effect is essentially permanent for practical purposes. Heparin (in green tops) works by a completely different mechanism—it enhances a natural anticoagulant in the body rather than binding calcium at all. Sodium citrate’s gentle, reversible chelation makes it ideal for coagulation testing because laboratories can add calcium back to the sample and measure exactly how the clotting cascade functions.
The Critical 9:1 Ratio
Light blue tubes collect blood at a precise 9:1 ratio—nine parts blood to one part sodium citrate. This ratio is the result of decades of research determining the exact balance needed for accurate testing.
Too much citrate (underfilled tube) binds more calcium than necessary, making tests take longer to clot and falsely suggesting coagulation problems. Too little citrate (overfilled tube) can’t fully anticoagulate the blood, causing micro-clots that make the specimen unsuitable.
Light blue tubes must be filled to the line—it’s a requirement, not a suggestion. Short-draws are one of the most common rejection reasons. Even a 10-15% underfill can significantly affect PT/INR results. For difficult draws, many facilities keep partial-draw/short-draw citrate tubes (smaller tubes with proportionally less citrate) for pediatric patients or slow-flowing venipunctures.
Why Contamination Is So Dangerous
EDTA (from lavender tubes) chelates calcium even more strongly than citrate. If your needle carries even a tiny amount of EDTA into the light blue tube, it locks up additional calcium beyond what citrate already bound, causing falsely prolonged clotting times. A normal patient might appear to have a coagulation disorder.
Heparin contamination is equally problematic. Even trace amounts from a green top can falsely prolong PTT results by activating antithrombin, which inhibits clotting factors.
Drawing red or gold tops (serum tubes with clot activators) before light blue tubes is also dangerous. Clot activators transferred via the needle can trigger premature clotting, consuming factors before testing—leading to falsely prolonged results.
Here’s the insidious part: laboratories can’t detect mild to moderate contamination. It just looks like the patient has a coagulation abnormality. The lab reports the results, the physician acts on them, and the patient gets treated based on false information. Studies document real cases of inappropriate anticoagulation therapy, delayed surgeries, and unnecessary workups—all from preventable collection errors.
Modern Standards and Best Practices
Current CLSI guidelines specify for the sodium citrate tube:
Position: Second, immediately after blood cultures (or first if no blood cultures)
Fill volume: Must reach the marked line (90-100% of capacity)
Mixing: Gently invert only 3-4 times immediately after collection. Over-inverting activates platelets, causing them to release substances that interfere with testing. Vigorous mixing also causes hemolysis, which affects results. Mix just enough to distribute citrate evenly without mechanically stressing blood cells.
Discard tube: If you are collecting blood from a butterfly or an IV and need to use a discard tube to get rid of excess air or contamination, you cannot use a serum tube (red/gold). The tube needs to be a true non-additive (usually with a white cap), or, what is probably easiest is to just use another sodium citrate tube. A syringe is also acceptable.
The Connection to Patient Care
Precision in phlebotomy has direct consequences for patient safety. A patient on warfarin for atrial fibrillation needs regular INR monitoring. Too low means stroke risk; too high means hemorrhage risk. The difference can be tenths of a point. If you contaminate the light blue tube with EDTA, the INR appears falsely elevated. The physician might reduce warfarin—putting the patient at risk for stroke.
That’s why light blue tubes come second, why the 9:1 ratio matters, and why we don’t draw other tubes first. It’s about generating reliable results physicians can trust to make safe treatment decisions.
Looking Ahead
Light blue tubes exemplify why the order of draw isn’t arbitrary. Sodium citrate’s reversible calcium binding makes it perfect for coagulation studies—but only if the specimen isn’t contaminated.
Next week, we’ll explore red top tubes and the evolution of clot activators—from plain glass tubes to modern tubes with silica particles or thrombin. We’ll discover why tubes without anticoagulants come before tubes with stronger anticoagulants, and how serum collection has evolved.
Have you seen people frequently draw light blue tubes out of order? Share your experiences with us.
Previously in the series: Blood Culture Bottles - What is SPS and why are the bottles always first?
Next in the series: Red Top Tubes - The Evolution of Clot Activators and Serum Collection
Great article on the Lt. Blue tube. Nice to see real information on what all phlebotomists should know.
Icing citrate tubes once was required, did you know? Fun fact.
Currently, trying to get the fill to the line on a citrate when it's inverted in its holder, seeing through a holder, taking out a tube to check? Well I lost a valuable vessel in the process 2 techs struggled to complete last weekend, sigh.
I have to say precise vacuum in that tube could help us a whole lot as our BD manufactured tubes can easily overfill.
I'll close with my experience with the quality of manufactured tubes today? I've never had to check more carefully, I don't know how quality control slid so hard, but in my opinion perhaps Kimble-Terumo is better?