We Need to Talk About Red Tubes

Clearing up the dangerous misconceptions about additives and order of draw

A while back, I was invited by Tina Dihle, the northern Colorado district representative of ASCLS, to speak at the Colorado ASCLS fall seminar. I was excited to accept. It’s been a while since I was a speaker at a conference, and while public speaking used to terrify me when I was younger, I now quite enjoy the opportunity to share my knowledge and meet new people. Plus, I love a good road trip.

That seminar took place just a couple of weekends ago, and I had a wonderful time. I drove from northern Utah to northern Colorado. Tina showed me all around Fort Collins, and I got to see a little of the Colorado State University campus where she worked for many years. What a beautiful place—if you’ve never been there, it’s worth a visit for sure.

The conference took place in Longmont, about 45 minutes south of Fort Collins. I was able to meet an absolutely incredible group of women who are clearly passionate about what they do. The speakers were excellent, and I learned quite a lot about new blood culture technology and newborn blood typing.

My topic was preanalytical errors that can lead to injuries to patients, both physical (such as nerve injury) and diagnostic (altered test results). One of the things I discuss when I give presentations like this is how red top tubes are often used as discard tubes before light blue tubes. There’s a huge misconception that plain red top tubes have no additive, which just isn’t true. Every time I give this presentation, there’s surprise among the attendees when they learn red top tubes can cause problems (though I think this awesome group was pretty aware of the problems I am about to explain).

So let’s talk about what’s actually going on with red tubes, why the confusion exists, and what you really need to know about order of draw.

The Misconception

Here’s what a lot of people believe: “Red tubes have no additive, so they’re safe to use as a discard tube.”

If I had a dollar for every time I’ve heard some version of this, I could retire and spend my days doing something less stressful—like defusing bombs.

The problem is, this statement is only partially true in very specific circumstances, and those nuances matter. A lot.

Just this week, I was scrolling through Reddit (because apparently, that’s what I do when I should be doing other things), and I came across a thread that proved this misconception is alive and well. People were confidently stating that red tubes—both glass AND plastic—can be used as discard tubes before drawing a light blue coagulation tube when using a butterfly set.

And I’m over here like... deep breath... no. No, they can’t.

Let’s break down why.

Glass Red Tubes vs. Plastic Red Tubes: They’re Not the Same

The Old School Glass Red Tube

Back in the day, red glass tubes were the standard for serum collection. And yes, technically speaking, they contain no chemical additive. There’s nothing added to that tube—no gel separator, no clot activator powder, nothing.

But—and this is a huge but—the glass itself acts as a clot activator.

BD Vacutainer^® Serum Tubes

When blood comes into contact with glass, it activates Factor XII and kicks off the intrinsic clotting pathway. The glass surface does the work that additives do in modern plastic tubes. So while there’s no additive in the technical sense, that tube is absolutely activating clotting the moment blood touches it.

The Modern Plastic Red Tube

Now, plastic red tubes are a different story entirely. Plastic is inert—it doesn’t activate clotting on its own. That’s why manufacturers had to add clot activators like silica particles to their plastic red tubes to replace what the glass used to do naturally. The tube might look plain, but functionally, there’s something in there making that blood clot very thoroughly. BD and Greiner—the tubes you’re most likely using in the US—both include these additives in their plastic red tubes.

VACUETTE® TUBE 2 ml CAT Serum Clot Activator

Now, do some manufacturers make plain plastic red tubes with absolutely no additive? Yes— they are rare, but you can find them from smaller manufacturers, mostly overseas, and from some veterinary suppliers. But in typical US clinical settings with major brands? If you’re grabbing a plastic red tube off your cart, it almost certainly has a clot activator in it.

How Did We Get Here?

Here’s where things get messy. Back in the day, people learned that red glass tubes have no additive. They also learned that red tubes came BEFORE light blue tubes in the order of draw—which was the standard until 2003.

Why was that the old order? Because of a myth about tissue thromboplastin.

For years, everyone believed that the trauma of a venipuncture caused tissue thromboplastin from the injured tissue to accumulate in the needle. The theory was that if you drew a coagulation tube first, this tissue thromboplastin would contaminate the sample and mess up your PT and aPTT results. So the solution was to either use a discard tube first, or draw other tubes (like red tubes) before the light blue.

But here’s the thing: multiple studies in the late 1990s proved this wasn’t actually happening. Tissue thromboplastin from the needle stick didn’t affect coagulation results when the citrate tube was drawn first. The myth was busted. In 1998, CLSI even revoked the discard tube requirement for straight needle draws because it simply wasn’t necessary.

Meanwhile, plastic tubes were gradually replacing glass tubes throughout the 1990s and early 2000s. And this created a NEW problem that the old order of draw couldn’t handle.

Remember, plastic doesn’t activate clotting like glass does. So manufacturers started adding clot activators (like silica) to plastic serum tubes. And studies started showing that these clot activators COULD carry over into the next tube and contaminate coagulation samples. In 2012, a study by Fukugawa and colleagues tested the effects of different clot activators on coagulation results. They found that when citrate tubes were drawn after tubes containing clot activators (including tubes with glass particles combined with other activators), PT and PT-INR results were falsely shortened—small changes, but statistically significant and clinically relevant.

Importantly, we don’t have studies that specifically tested plain glass red tubes alone for carryover effects. The Fukugawa study looked at glass particles in combination with other additives. So while we know glass activates clotting through contact activation, we don’t have direct evidence showing how much that affects citrate tubes in practice.

So you had this perfect storm:

• People trained on glass tubes (where “no additive” was technically true)

• A myth about tissue thromboplastin that had just been disproven (making it safe to draw citrate tubes first)

• A major order of draw change in 2003 (moving serum tubes AFTER light blue tubes)

• AND a switch to plastic tubes that DO contain clot activators (making the new order essential)

The 2003 change happened specifically because we now understood that clot activator carryover was a real threat to coagulation testing—not tissue thromboplastin. Moving serum tubes after light blue tubes solved the problem.

Many people however, thought that red plastic tubes were the exact same thing as the red glass tubes, and that besides being made of plastic that there were no other changes. They never learned that the plastic tubes have additives to make blood clot thoroughly.

Many people think that the blood just clots on its own, (which it does, but not always very well). The silica ensures you get a fast, thorough clot, which is critical for good serum separation. When the clot forms quickly and completely, you get a clean serum sample on top that’s free of fibrin strands and cellular material. This is also why the glass tubes worked so well for so long.

But, the misconceptions from that era have stuck around like gum on a shoe. New phlebotomists get trained by veteran phlebotomists who learned the old rules and never got the update about the nuances. Some still think red tubes can go before light blues. Some think “no additive” means “no clotting activation.” The misinformation spreads, and it’s incredibly difficult to correct.

What Does Current CLSI Guidance Say?

This year (2025), CLSI released PRE02, which replaced the long-standing GP41 standard. Here’s what they recommend for order of draw (hint, it’s the same as it has been since 2003):

1. Blood culture tube or bottle

2. Sodium citrate tube (light blue)

3. Serum tubes (red, gold, speckled)

4. Heparin tubes (green)

5. EDTA tubes (lavender, pink)

6. Glycolytic inhibitor tubes (gray)

CLSI states that this order is “recommended for both glass and plastic venous collection tubes… Its purpose is to avoid possible test result error due to additive carryover.”

But here’s where it gets interesting: CLSI also includes a note that states “Plastic serum tubes containing a clot activator can cause interference in coagulation testing. Only blood culture tubes, glass nonadditive serum tubes, or plastic serum tubes without a clot activator may be collected before the coagulation tube.”

So CLSI makes an exception for glass nonadditive red tubes—technically, they CAN be drawn before light blue tubes.

But should they?

What About Plain Red Tubes with No Additive?

“So,” you might be thinking, “CLSI says glass nonadditive tubes CAN be drawn before citrate tubes. So what’s the problem?”

Here’s my take: just because something is technically allowed doesn’t mean it’s best practice.

Yes, CLSI makes an exception for glass nonadditive tubes in their order of draw guidance. But consider these points:

Glass DOES activate clotting: Even without a chemical additive, glass surfaces activate Factor XII and the intrinsic clotting pathway. Blood in that tube is clotting.

We don’t have the data: The studies showing clot activator contamination tested glass particles combined with other additives. We don’t have good studies that specifically looked at plain glass red tubes alone. That doesn’t mean they’re safe—it means the question hasn’t been thoroughly studied.

Why risk it?: When the stakes are accurate coagulation results that directly affect patient care (anticoagulation management, pre-op screening, clotting disorder diagnosis), why would we rely on a technical exception when the science suggests there could be a problem?

Most facilities use plastic anyway: For the majority of facilities using plastic tubes, this isn’t even relevant. And if you’re using plastic red tubes from major manufacturers, they definitely contain clot activators.

My position is simple: err on the side of caution. Draw ALL serum tubes—glass or plastic—after your citrate tubes. Don’t use red tubes as discard tubes.

So What’s the Problem with Using Red Tubes Before Light Blue Tubes?

Okay, let’s get to the heart of why this matters for order of draw.

Whether you’re using a glass red tube or a plastic red tube with additives, blood in that tube is actively clotting. And when you draw that tube first—especially with a butterfly setup—you’ve got blood sitting in your tube needle that’s been exposed to a clot-activating surface.

When you then push your light blue tube onto the back of the butterfly needle, guess what happens? That blood that has begun to clot can carry over into your coagulation tube.

Even microdroplets matter. PT, PTT, INR—these tests are sensitive. Contaminating your light blue tube with blood that’s already started the clotting process will give you inaccurate results. And inaccurate coagulation results can lead to really bad patient outcomes.

Let Me Paint You a Picture

Here’s what this looks like in real life:

Mrs. Johnson comes in for her routine labs. She’s been on warfarin (Coumadin) for atrial fibrillation for the past five years, and she gets her PT/INR checked monthly to make sure her blood isn’t too thick (clotting risk) or too thin (bleeding risk). Her doctor also ordered a comprehensive metabolic panel.

The phlebotomist is having a busy morning. They’re using a butterfly set because Mrs. Johnson has small veins. Without thinking much about it, they draw the red tube first to clear the air from the line, then follow with the light blue for the PT/INR. She knows the light blue tube must be full, and she falsely believes drawing the red tube first is harmless since it contains no additive.

The red tube looks fine. The light blue tube looks fine. Everything seems normal.

But here’s what actually happened: Blood in that red tube immediately started clotting because of the silica clot activator. Some of that partially clotted blood—maybe just microdroplets—carried over into the light blue tube through the butterfly tubing.

The lab runs the PT/INR. It comes back at 1.8.

Mrs. Johnson’s therapeutic range is supposed to be 2.0-3.0. A result of 1.8 looks subtherapeutic—like her blood is clotting too easily and she’s at risk for a stroke. So her doctor increases her warfarin dose.

Except Mrs. Johnson’s actual INR was fine. The low result was an artifact from contamination. Now she’s getting a higher dose of warfarin than she needs, putting her at risk for bleeding complications. A few weeks after her increased dose in medication she falls and hits her head. She has intracranial bleeding and develops a subdural hematoma requiring emergency surgery.

Her new blood tests show her blood is 3.8, dangerously high and well above her therapeutic range. What started as a routine monthly blood draw ended with a patient in the ICU, all because someone thought a red tube was acceptable to draw before the light blue tube.

This is not a hypothetical. This kind of thing happens. And it happens because someone didn’t understand that red tubes can mess up coagulation results. No one would have any way of knowing that this mistake occurred unless an event happened that prompted investigation. And you probably know the saying, “safety standards are written in blood,” because unfortunately that is how changes are often made.

“But What About Clearing the Air from the Tubing?”

This is where the Reddit argument was happening, and I hear this one constantly.

The logic goes like this: “When you use a butterfly, there’s air in the tubing. You need to draw a discard tube to clear the air before drawing the light blue. Since red tubes have no additive, they’re perfect for this.”

And, as explained above, this is not correct.

The correct approach? Use a proper discard tube:

• A true non-additive tube (white-topped tubes that say “non-additive” and aren’t made of contact-activating glass), OR

• An extra light blue citrate tube as your discard (this is my favorite option)

  

Both of these options clear the air from the tubing without exposing the blood to clot activators or contact-activating surfaces. Then your actual test tube gets blood that hasn’t been compromised.

Using a red tube—any red tube—defeats the entire purpose of protecting the integrity of your coagulation sample.

The Bottom Line

When using a butterfly and you need a discard tube: Do NOT use a red tube (glass or plastic). Use a non-additive or additional light blue.

The point of a discard tube when using a butterfly is to fill the dead space in the tubing so your citrate tube fills properly and maintains the correct blood-to-anticoagulant ratio. You don’t want to fill that space with blood that’s been exposed to clot activators.

My recommendation: Draw ALL serum tubes—glass or plastic—after your light blue citrate tubes. Don’t rely on the technical exception CLSI makes for glass tubes. When it comes to coagulation testing that affects patient anticoagulation management, err on the side of caution.

I know this might seem like I’m being picky about details. But here’s the thing: phlebotomy is built on details. The difference between a good draw and a bad draw, between accurate results and inaccurate results, often comes down to understanding these nuances.

When you understand why the guidelines exist—not just what the guidelines are—you make better decisions in the moment. You know when you can adapt and when you need to stick to the rules.

And you definitely don’t end up confidently giving bad advice on Reddit.

Trust me, your patients’ coagulation results will thank you.

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Is this a problem you have run into in your experience in phlebotomy?

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A big thanks to Tina and the wonderful lab scientists in northern Colorado who were so welcoming to me. I hope you will have me back sometime :)