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The Order of Draw Series: Part Five - EDTA Tubes

This week, we're examining EDTA tubes and discovering why the most commonly drawn tube in many facilities must wait until the fifth position despite its frequent use

by Shanise Keith

I have said – and when I say “said”, I mean I have ranted about this before, but when someone is not aware of the order of draw, they tend to collect the lavender tube first. It is the most commonly collected tube out of them all. Almost every patient will need a lavender tube collected every single time they get their blood drawn, and it becomes a staple. Someone who doesn’t understand the order of draw usually puts this tube first because every patient needs it.

The lavender EDTA tube is one of the most problematic tubes, which is why it comes so late in the order of draw. Any of you watching HBO Max’s The Pitt probably noticed that in the most recent episode (season 2, episode 4), a new nurse was being instructed by the charge nurse on how to do a blood draw. The venipuncture and technique were terrible, and the charge nurse’s instructions weren’t great (not surprising). But, of course, the order of draw was also wrong. The nurse drew the lavender tube before the serum tube, which had me yelling at the TV, (and you probably were too). Thankfully, she dropped the serum tube which also happened to be glass, and it was broken. A redraw of the red tube would fix the problem that they were not even aware of.

Medical shows like this never get the phlebotomy right, but I was particularly disappointed in this show, because it claims to be the most medically accurate. Having worked in the ER for several years myself I can tell you that a lot of it is pretty spot on, but such a simple thing as phlebotomy and the order of draw gets missed? They couldn’t have consulted with an experienced phlebotomist for the few episodes they may have where we see the phlebotomy procedure? It’s a bummer for sure, but maybe they will do better in future episodes.

Why EDTA Tubes Come Fifth

After blood cultures, light blue coagulation tubes, serum tubes, and green heparin tubes, the order of draw brings us to EDTA tubes—most commonly recognized by their lavender or purple tops. These tubes hold the fifth position for a critical reason: EDTA is an extremely effective calcium chelator, and even trace contamination of other tubes can cause serious preanalytical errors.

EDTA binds calcium with remarkable tenacity. While sodium citrate in light blue tubes also chelates calcium, citrate binds reversibly—the laboratory can add calcium back to reverse the anticoagulation when needed for specific tests. EDTA, however, binds calcium essentially permanently for practical purposes. This makes EDTA contamination of other tubes particularly problematic because there’s no easy way to undo it.

EDTA tubes come fifth in the order of draw to ensure that this powerful calcium chelator cannot contaminate tubes drawn before it. By the time we reach the lavender top, all tubes sensitive to calcium chelation have already been collected safely.

The History of EDTA in Laboratory Medicine

The story of EDTA begins in 1935, when German chemist Ferdinand Munz first synthesized ethylenediaminetetraacetic acid (say that five times fast). Originally developed for industrial applications—particularly for removing calcium deposits from boilers and for water softening—EDTA’s potential for biological applications wasn’t immediately obvious.

In the early 20th century, laboratory hematology faced a significant challenge. Blood needed to be kept liquid for cell counting and microscopic examination, but the available anticoagulants all had serious drawbacks. Oxalate salts, commonly used at the time, caused slight hemolysis (rupture of red blood cells) and could distort cell morphology. Heparin was expensive and sometimes caused clumping of white blood cells. Sodium citrate required significant dilution of the blood sample, which affected cell counts.

In the 1940s and 1950s, researchers began exploring EDTA as a biological anticoagulant. They discovered something remarkable: EDTA preserved blood cell morphology better than any anticoagulant available. Red blood cells maintained their normal shape and size. White blood cells remained intact for examination. Platelets didn’t clump or aggregate. The cells in an EDTA tube looked remarkably similar to cells in fresh, flowing blood.

By the 1960s, EDTA had become the anticoagulant of choice for hematology. The development of automated cell counters in the 1960s and 1970s accelerated this adoption—these instruments needed specimens where cells remained stable and well-preserved, and EDTA delivered exactly that. Today, EDTA is the universally accepted anticoagulant for complete blood counts and blood bank testing worldwide.

How EDTA Works: The Chemistry of Chelation

EDTA prevents blood clotting through a process called chelation—the chemical binding of metal ions. The EDTA molecule has a unique structure with multiple binding sites that can wrap around and trap metal ions, particularly calcium.

Think of EDTA like a molecular claw with four “fingers” (the four acetic acid groups in its structure) that can grab onto a calcium ion and hold it tightly from multiple angles at once. Once EDTA grabs calcium, it doesn’t let go easily. This binding is so stable that for practical purposes, the calcium is essentially removed from circulation in the blood sample.

Here’s why this matters for preventing clotting: calcium is absolutely essential for the coagulation cascade. Multiple steps in the clotting process require calcium ions to function. Calcium helps activate various clotting factors, allows platelets to aggregate properly, and enables the formation of fibrin—the protein mesh that forms the structure of a blood clot.

When EDTA removes calcium from the equation, the entire clotting cascade shuts down. It’s like removing a critical component from an engine—without calcium, the system simply cannot run. The blood remains liquid indefinitely because clotting cannot proceed.

Importantly, EDTA chelates calcium without damaging blood cells. The process is gentle. Red blood cells maintain their normal biconcave disc shape. White blood cell nuclei remain intact. Platelet structure is preserved. This is why EDTA became the gold standard for hematology—it stops clotting while preserving the very cells we need to examine.

EDTA is added to collection tubes as K2EDTA (dipotassium EDTA), a potassium salt that makes the EDTA water-soluble. Historically, K3EDTA (tripotassium EDTA) was also used, but it has been largely phased out because the higher potassium concentration caused a slightly more osmotic effect on red blood cells, affecting cell volume measurements. K3EDTA tubes are rarely seen in modern practice.

The fact that EDTA exists as a potassium salt is critical to understanding contamination issues—when EDTA contaminates other tubes, it introduces additional potassium, falsely elevating potassium results.

The Rainbow of EDTA Tubes: Lavender, Pink, Tan, and Royal Blue

While we commonly refer to “lavender top” EDTA tubes, EDTA actually comes in several different colored tubes, each designated for specific testing purposes:

All of these tubes contain EDTA and serve the same fundamental purpose—chelating calcium to prevent clotting—but the different colors help ensure specimens are sent to the correct laboratory department and processed appropriately. When drawing multiple EDTA tubes, they all occupy the same position in the order of draw (fifth), regardless of their color.

The Critical Importance of Proper Fill Volume

EDTA tubes require more precise filling than almost any other tube type (light blue being the exception). The fill line marked on the tube isn’t just a suggestion—it’s critical for accurate results.

EDTA tubes are designed for a specific blood-to-anticoagulant ratio. Most EDTA tubes are designed to collect 1.8 mL of blood, though larger and smaller volumes are available. When filled to the proper level, the ratio of blood to EDTA is optimized for cell preservation and accurate testing.

Overfilling an EDTA Tube: If you collect too much blood—say, 2.5 mL in a tube designed for 1.8 mL—you dilute the EDTA. There may not be enough EDTA present to fully chelate all the calcium in the larger blood volume. This can allow micro-clots to form. Even tiny clots invisible to the naked eye can cause problems. Automated cell counters cannot count cells in clotted specimens, and the specimen will be rejected. The patient needs to be redrawn.

Underfilling an EDTA Tube: If you collect too little blood—say, 1.0 mL in a tube designed for 1.8 mL—you’ve created a specimen with too much EDTA relative to the blood volume. Excess EDTA creates a strong osmotic gradient that draws water out of cells, causing them to shrink. This shrinkage falsely lowers the mean corpuscular volume (MCV) and can affect the hematocrit. Additionally, excess EDTA can distort cell morphology and interfere with some automated cell counter measurements.

For newer phlebotomists, here’s the practical takeaway: Fill EDTA tubes to the marked line (sometimes a square, or line on the label). Use the correct tube size for the amount of blood you can obtain. If you’re struggling with a difficult draw and can only get a small amount of blood, use a pediatric or partial-draw EDTA tube rather than putting a small amount of blood in a full-size tube.

For experienced phlebotomists, you’ve probably gotten that call from the lab: “Specimen clotted in the EDTA tube.” Often, that’s an overfilled tube where there wasn’t enough EDTA for the blood volume. But there’s another cause you’ve likely encountered: slow blood flow allowing clotting to begin before the blood reaches the EDTA.

When Blood Clots Before Reaching the EDTA

Here’s a critical point that deserves emphasis: EDTA cannot reverse coagulation once it has started. EDTA is a preventive anticoagulant—it stops clotting by chelating calcium before the coagulation cascade can proceed. But once clotting factors have been activated and fibrin strands have started forming, EDTA cannot undo that process.

This becomes a real issue with slow blood flow, particularly common when using butterfly needles, drawing from small or fragile veins, or managing partial obstructions. When blood flow is sluggish, blood may sit in the tubing or needle for several seconds before reaching the collection tube. During that time, the coagulation cascade can begin.

Blood begins clotting within seconds of leaving the vein—that’s exactly what it’s designed to do. If blood flow through your butterfly line is very slow, tiny clots may form in the tubing before the blood ever reaches the EDTA in the tube. Even if you collect the correct volume and mix properly, those micro-clots are already present. The EDTA prevents additional clotting, but it can’t dissolve the clots that have already formed.

The laboratory’s automated cell counter cannot differentiate cells in specimens containing even microscopic clots. The instrument flags the specimen as clotted and rejects it. You get the call: “Specimen clotted in EDTA tube”—and you know you filled it to the line and mixed it properly, so what’s the deal? It was likely already clotting as it was being collected.

Practical strategies for slow-flow situations:

This is one of those situations where perfect technique still sometimes results in specimen rejection—not because you did anything wrong, but because the patient’s blood flow was too slow to prevent coagulation initiation before EDTA contact. It’s frustrating, but understanding the chemistry helps explain why it happens.

Clinical Consequences: When Order of Draw Violations Affect Patient Care

Let’s walk through some scenarios that illustrate why EDTA’s position in the order of draw matters:

Scenario 1: Masked Hypokalemia from EDTA Contamination

A patient is scheduled for elective surgery the following morning. Pre-operative labs are ordered, including a comprehensive metabolic panel (CMP). The phlebotomist draws blood in this order: lavender top for a CBC, then gold top for the CMP.

EDTA from the lavender tube contaminates the gold top. The patient’s true potassium is 2.8 mEq/L—significant hypokalemia that should delay surgery until corrected. However, the contaminating K2EDTA adds potassium to the specimen, and the result comes back at 4.2 mEq/L—appearing completely normal. The patient is cleared for surgery.

During the procedure, under the stress of anesthesia, surgical manipulation, and fluid shifts, the patient’s undetected hypokalemia triggers a cardiac arrhythmia. The patient’s heart suddenly stops. The surgical team begins resuscitation—CPR, defibrillation, emergency medications.

During the code, a stat basic metabolic panel is drawn using proper technique. The potassium comes back at 2.7 mEq/L—critical hypokalemia. The team administers IV potassium replacement while continuing resuscitation efforts. The patient is eventually stabilized, but they’ve experienced a preventable cardiac arrest that could have been avoided if the pre-operative hypokalemia had been detected and corrected before surgery.

The EDTA contamination didn’t create a false abnormality—it masked a real, life-threatening one. The added potassium from the K2EDTA salt falsely normalized the result, making a critically low potassium appear normal and allowing a high-risk patient to proceed to surgery.

Scenario 2: Unnecessary Repeat Draws

A phlebotomist draws tubes in the wrong order, collecting the lavender EDTA tube before the green heparin tube for chemistry testing. The magnesium result comes back critically low at 0.8 mg/dL (normal range 1.7-2.2).

The physician orders an immediate redraw to confirm before starting magnesium replacement. The patient must be stuck again. The repeat draw—done in the correct order this time—shows a normal magnesium of 1.9 mg/dL. The first result was wrong due to EDTA contamination.

These aren’t hypothetical scenarios. These are real preanalytical errors that occur in laboratories every day when the order of draw isn’t followed. EDTA contamination creates a particularly dangerous combination of false results—falsely prolonged coagulation times, falsely low calcium and magnesium, and falsely elevated potassium—all of which can trigger inappropriate, potentially harmful interventions.

Modern Standards and Best Practices

The Clinical and Laboratory Standards Institute (CLSI) publishes the PRE02 standard, “Collection of Diagnostic Venous Blood Specimens,” which specifies the order of draw and handling requirements for EDTA tubes.

Key CLSI recommendations for EDTA tubes:

The mixing technique deserves emphasis. After you finish drawing the EDTA tube, immediately invert it 8-10 times. This ensures the EDTA coating the tube walls mixes thoroughly with the blood. Incomplete mixing can allow micro-clots to form even in an EDTA tube. Invert gently—you’re mixing, not shaking a cocktail. Vigorous shaking can rupture red blood cells and cause hemolysis.

For storage, EDTA specimens should be kept at room temperature. Refrigeration can activate platelets and affect some cell counts. Some tests (like flow cytometry) require processing within specific timeframes, so check your laboratory’s specific requirements.

Why EDTA Revolutionized Hematology

Before EDTA became standard, examining blood cells under a microscope was challenging. Cells would deteriorate rapidly. Automated cell counting was impossible because cells clumped, lysed, or changed shape in older anticoagulants.

EDTA changed everything. It provided laboratories with specimens where cells remained stable for hours. This stability enabled the development of automated hematology analyzers—instruments that could count millions of cells with precision and generate comprehensive CBCs in minutes. Modern hematology, as we know it today, wouldn’t exist without EDTA.

That lavender top tube represents more than just a blood collection device. It represents decades of scientific advancement in understanding how to preserve human cells, how to prevent clotting without damaging specimens, and how to generate accurate laboratory results that guide patient care.

Every time we draw an EDTA tube in the correct position in the order of draw, we’re protecting the accuracy of those results and, ultimately, protecting our patients.

Next week, we’ll explore gray top tubes with sodium fluoride and potassium oxalate—the specialized tubes for glucose testing. We’ll discover why these tubes come last in the order of draw, trace the history of how we learned to preserve glucose in blood specimens, understand the dual anticoagulant system these tubes employ, and see why sodium fluoride contamination of other tubes creates some of the most serious preanalytical errors in clinical chemistry.

The order of draw is a patient safety protocol built on decades of evidence about how tube additives interact. Understanding why lavender tops sit where they do in the sequence—and what goes wrong when we deviate from that sequence—makes us better collectors, better educators, and better advocates for accurate laboratory results.

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