The Complete Phlebotomy Guide: What Every Student and Working Phlebotomist Needs to Know
Written by Nancy L. Kimmel, MSN, FNP — Founder of Phlebotomy Career Training, Family Nurse Practitioner, and phlebotomy educator with over 17 years of clinical and teaching experience.
At Phlebotomy Career Training, we consider ourselves pioneers in online phlebotomy education. For 16 years, we have been training students across the country — not just to pass a certification exam, but to understand the why behind every step of the phlebotomy process. This page is dedicated to exactly that: giving students, working phlebotomists, nurses, and anyone curious about the science of blood collection the real, evidence-based knowledge that separates a competent phlebotomist from a great one.
Knowledge should be accessible. That is the philosophy we were built on, and that is why this guide is completely free.
Why Evidence-Based Phlebotomy Matters
One of the most important things to understand about phlebotomy — and about medicine in general — is that correct technique is not simply a matter of tradition. For too long, healthcare workers did things a certain way because that is how they were taught, and their teachers were taught the same way, going back generations. That is not science. That is habit.
Consider hand washing. It seems obvious today, but there was a time when surgeons performed operations without washing their hands at all. Patients died from hospital-acquired infections at alarming rates. It was not until scientists like Louis Pasteur demonstrated that bacteria multiply in stagnant conditions that the medical community began to understand the critical importance of hand hygiene. The science changed the practice — and lives were saved.
The same principle applies to phlebotomy. As a Family Nurse Practitioner, I can tell you with certainty: there is a mode, a method, and a scientific reason for every step in the phlebotomy process. At Phlebotomy Career Training, we teach phlebotomy the way it should be taught — grounded in evidence-based science, not outdated habit.
Understanding Blood: The Foundation of Phlebotomy
Before a phlebotomist can truly master the order of draw or understand the difference between collection tubes, they need to understand what blood actually is. This is where most phlebotomy training falls short — and where we begin.
Blood is a tissue. Just like your skin (epidermis), your bones, and your heart muscle are all tissues, blood is a specialized tissue that circulates throughout your body. Every tissue in the human body is specialized — cardiac myocytes make up the heart muscle, columnar and squamous epithelium line the kidneys’ nephrons — and blood is no different. It is a living, dynamic tissue with a very specific job.
Think of blood like a freight train with four parallel tracks, all moving simultaneously. It carries everything your body needs to survive: red blood cells, white blood cells, platelets, clotting factors, hormones, vitamins, electrolytes, antibodies, and water. Blood has velocity and viscosity. As it moves through arteries, veins, arterioles, venules, and capillaries, its speed and pressure change according to the laws of fluid dynamics.
Venous Blood vs. Arterial Blood
This is a foundational concept that every phlebotomist must understand. Venous blood and arterial blood are not the same.
Veins carry deoxygenated blood back toward the heart. From the heart, that blood travels to the lungs to pick up oxygen, returns to the left side of the heart, and is then pumped out to the rest of the body through the arteries. This means venous blood has significantly less oxygen than arterial blood — and that difference is visible. Venous blood is a darker, deeper red. Arterial blood is bright red, almost orange.
Veins are also more superficial (closer to the surface of the skin) than arteries, which is why phlebotomists draw from veins and not arteries. If a phlebotomist draws blood and it comes out bright red and pulsing, that is a serious problem — an artery has been punctured, and immediate action is required.
Phlebotomists draw from veins. Always. Understanding why is not just academic — it is a matter of patient safety.
Serum vs. Plasma: The Concept That Trips Up Even Experienced Phlebotomists
After 17 years in this field, I can tell you with confidence: the single hardest concept for phlebotomy students — and even for many working phlebotomists — is understanding the difference between serum and plasma, and why it matters for tube selection. Let us break it down clearly, once and for all.
What Happens When Blood Sits in a Plain Test Tube?
Imagine drawing venous blood and placing it into a completely plain glass test tube — no additives, no anticoagulants, nothing. Now set it on a counter and let gravity do its work.
Over time, the components of blood will separate according to their density, just like oil and vinegar separate in a salad dressing. What you will see is this:
- A beautiful amber layer on top — this is your serum.
- A dark, deep red layer on the bottom — these are your formed elements: red blood cells, white blood cells, platelets, and clotting factors.
Serum is everything in the blood except the formed elements. It contains hormones, vitamins, electrolytes, enzymes, antibodies — everything that floats in the liquid portion of blood once the cells and clotting factors have settled out. Serum chemistry is one of the most commonly ordered panels in clinical medicine, and it is tested using serum separator tubes.
What Is Thixotropic Gel — and Why Does It Exist?
Now let us run the same experiment, but this time the test tube has a small amount of a substance at the bottom that looks like thick, gooey honey. That substance is called thixotropic gel.
Thixotropic gel was engineered by chemists with a very specific purpose. Its density is carefully calibrated to be:
- Less dense than the formed elements (so it floats above them)
- More dense than the serum (so it sinks below the serum)
When the tube is placed in a centrifuge and spun, the thixotropic gel migrates to exactly the right position — forming a physical barrier between the serum on top and the formed elements on the bottom. This barrier is not just clever chemistry. It is a practical solution to a real laboratory problem.
Without that barrier, when the medical laboratory technologist tries to pour off the serum, the formed elements mix back in. The sample is ruined. The patient has to be redrawn. Nobody wants that — not the patient, not the phlebotomist, and certainly not the lab.
With the thixotropic gel barrier in place, the lab tech can cleanly pour off the serum and run up to 14 different chemistry tests from a single tube. Those tests include:
| Test Category | Examples |
|---|---|
| Electrolytes | Sodium, Potassium, Chloride, CO? |
| Kidney Function | BUN (Blood Urea Nitrogen), Creatinine |
| Metabolic | Glucose, Alkaline Phosphatase (Alk Phos) |
| Liver Enzymes | AST (Aspartate Aminotransferase), ALT (Alanine Aminotransferase) |
| Proteins | Total Protein, Albumin |
| Calcium & Phosphorus | Calcium, Phosphorus |
The Serum Separator Tubes: More Than One, Same Purpose
Here is where it gets confusing — and where many phlebotomists get tripped up. There is not just one serum separator tube. There are several, and they all serve the same fundamental purpose. Here is a quick reference:
| Tube Color | Contains Thixotropic Gel? | Common Use |
|---|---|---|
| Gold (SST) | Yes | Most common serum chemistry panel |
| Bright Red | No (plain) | Medication drug levels, blood bank |
| Dark Red / Burnt Umber | Sometimes | Serum chemistry, varies by lab |
| Marble / Mottled Top | Yes | Serum chemistry (older designation) |
All of these are serum separator tubes. The color coding varies by manufacturer and institution, which is why it is critical to know your facility’s specific tube guide — but the underlying science is always the same.
The Order of Draw: Why It Matters More Than You Think
The order of draw is one of the most tested topics on phlebotomy certification exams — and one of the most misunderstood in real clinical practice. I have personally experienced the consequences of incorrect order of draw as a nurse practitioner drawing blood from PICC lines: using a heparin flush before drawing coagulation labs (PT, PTT) completely invalidated those results. It is not a theoretical problem. It happens every day in hospitals and clinics across the country.
The order of draw exists to prevent cross-contamination between tube additives. The standard is established by the Clinical and Laboratory Standards Institute (CLSI). Each tube contains different additives — anticoagulants, clot activators, preservatives — and if a small amount of one additive is carried over into the next tube on the needle, it can alter the test results dramatically.
The standard order of draw established by the Clinical and Laboratory Standards Institute (CLSI) is:
| Order | Tube Color | Additive | Common Tests |
|---|---|---|---|
| 1 | Yellow (Blood Culture) | SPS (Sodium Polyanethol Sulfonate) | Blood cultures |
| 2 | Light Blue | Sodium Citrate (anticoagulant) | PT, PTT, INR, coagulation studies |
| 3 | Gold / Red / Marble (SST) | Clot activator ± thixotropic gel | Serum chemistry (BMP, CMP, lipids, etc.) |
| 4 | Green | Heparin (anticoagulant) | Plasma chemistry, STAT tests |
| 5 | Lavender / Purple | EDTA (anticoagulant) | CBC, hematology, ESR, reticulocytes |
| 6 | Pink | EDTA | Blood bank, type & screen |
| 7 | Gray | Potassium Oxalate / Sodium Fluoride | Glucose, lactate |
Note that the order of draw can vary slightly between hospital settings and outpatient labs. Some outpatient labs use only serum separator tubes and have a simplified draw protocol. Always follow your facility’s specific guidelines — but understand the reason behind the order, not just the sequence.
Serum vs. Plasma: The Final Word
Now that you understand serum, let us complete the picture with plasma — because this is the comparison that appears on every phlebotomy certification exam.
Serum is obtained from blood that has been allowed to clot. The clotting process consumes the clotting factors and fibrinogen, leaving behind the liquid portion — the serum. Serum does not contain clotting factors.
Plasma is obtained from blood that has been prevented from clotting by an anticoagulant (such as heparin, EDTA, or sodium citrate). Because clotting never occurred, plasma still contains all the clotting factors and fibrinogen. Plasma is the complete liquid portion of blood.
| Serum | Plasma | |
|---|---|---|
| Clotting factors present? | No | Yes |
| Fibrinogen present? | No | Yes |
| Tube type | Gold, Red, Marble (SST) | Green (heparin), Lavender (EDTA), Light Blue (citrate) |
| Common tests | Chemistry panels, hormones, enzymes | CBC, coagulation studies, STAT chemistry |
If you can internalize this distinction — serum has no clotting factors, plasma does — you have mastered one of the most fundamental and most tested concepts in phlebotomy. Congratulations. You are already ahead of many working phlebotomists in the field.
Learn Phlebotomy the Right Way — With the Experts Who Wrote the Book
Everything on this page reflects the same evidence-based, science-first approach we bring to every course at Phlebotomy Career Training. For 16 years, we have been training phlebotomists who do not just pass their exams — they understand their craft. They know why the order of draw matters. They know what thixotropic gel does and why it was invented. They know the difference between serum and plasma without hesitation.
Our Online Phlebotomy Certification Course includes everything you need to earn your CPT (Certified Phlebotomy Technician) credential through NAPTP — including the exam fee. You do not pay separately for the exam. It is included when you enroll.
If this page gave you value — if it helped something click that never clicked before — imagine what a full course with video instruction, hands-on technique guidance, and expert support can do for your career. You can also explore our free phlebotomy study guide to continue building your knowledge.
Enroll in Our Online Phlebotomy Course — Exam Fee Included
Frequently Asked Questions About Phlebotomy
What is the order of draw in phlebotomy?
The order of draw is the standardized sequence in which blood collection tubes are filled during a venipuncture procedure. The correct order prevents cross-contamination of tube additives, which can alter lab results. The standard order is: blood culture tubes, light blue (coagulation), gold/red/SST (serum chemistry), green (heparin/plasma), lavender (EDTA/CBC), pink (blood bank), and gray (glucose/lactate).
What is the difference between serum and plasma?
Serum is the liquid portion of blood after it has clotted — it does not contain clotting factors or fibrinogen. Plasma is the liquid portion of blood that has been anticoagulated, so it retains clotting factors. Serum is collected in gold, red, or marble-top tubes; plasma is collected in green, lavender, or light blue tubes.
What is a serum separator tube (SST)?
A serum separator tube (SST) is a blood collection tube that contains a clot activator and, in most cases, thixotropic gel. The gel forms a physical barrier between the serum and the formed elements (red cells, white cells, platelets, clotting factors) after centrifugation, making it easier for laboratory technologists to access the serum for chemistry testing.
What is thixotropic gel in blood collection tubes?
Thixotropic gel is a chemically engineered substance placed at the bottom of serum separator tubes. Its density is calibrated to be greater than serum but less than the formed elements of blood. When the tube is centrifuged, the gel migrates to form a barrier between the serum layer and the cellular layer, preventing contamination when the serum is poured off for laboratory testing.
Is venous blood different from arterial blood?
Yes. Venous blood is deoxygenated — it is returning to the heart and lungs to pick up oxygen — and appears darker red. Arterial blood is oxygenated and appears bright red. Phlebotomists always draw from veins, not arteries. If bright red, pulsing blood is obtained during a venipuncture, it indicates an arterial puncture, which requires immediate intervention.
How long does it take to become a certified phlebotomist?
With Phlebotomy Career Training’s online program, students can complete their phlebotomy certification training and sit for the NAPTP CPT exam in as little as a few weeks. The exam fee is included with enrollment — no separate exam registration fees required.
What is the NAPTP CPT certification?
The NAPTP (National Association of Phlebotomy Training Professionals) CPT (Certified Phlebotomy Technician) is a nationally recognized phlebotomy certification. Phlebotomy Career Training is an authorized NAPTP training provider, and the CPT exam fee is included with course enrollment.