Order of Draw and Specimen Handling
Fill tubes in the correct order, match additives to tests and keep specimens valid from collection to the lab.
Table of contents
A perfect needle stick can still produce a wrong result. A trace of additive carried from one tube to the next can change a potassium or clotting result, and a doctor may treat a problem that does not exist. A tube that sits too long, gets too warm or carries the wrong label is just as bad. This guide covers what happens to blood from the moment it enters a tube until it reaches the lab. It follows the order of draw published by the Clinical and Laboratory Standards Institute (CLSI) in its PRE02 standard, which replaced GP41 in 2025. Your lab's written procedure comes first.
What You Will Be Able to Do
- Match tube colors to additives and common tests.
- Fill tubes in the correct order and explain why.
- Mix, label and fill tubes correctly.
- Collect blood cultures without contaminating them.
- Handle specimens that need ice, warmth or protection from light.
- Recognize the reasons a lab rejects a specimen.
Tube Colors and Additives
Each stopper color tells you what is inside the tube. Colors can differ slightly between makers, so read the label too.
| Stopper | Additive | What it does | Common tests |
|---|---|---|---|
| Blood culture bottle | Growth medium | Grows bacteria | Blood cultures |
| Light blue | Sodium citrate | Stops clotting by binding calcium | PT, PTT, INR, fibrinogen |
| Red | None or clot activator | Lets blood clot for serum | Chemistry, drug levels |
| Gold | Clot activator and gel | Separates serum | Chemistry, thyroid, hepatitis |
| Green | Lithium or sodium heparin | Stops clotting | STAT chemistry, ammonia |
| Lavender | EDTA | Stops clotting and preserves cells | CBC, hemoglobin A1c, blood type |
| Pink | EDTA | Stops clotting | Blood bank tests, crossmatch |
| Gray | Sodium fluoride and potassium oxalate | Stops cells from using up glucose | Glucose, blood alcohol |
| Yellow | SPS or ACD | SPS for cultures, ACD for cell studies | Some cultures, HLA typing |
| Royal blue | EDTA or none, free of trace metals | Avoids metal contamination | Lead, trace elements |
The Order of Draw
Blood on the needle can carry additive from one tube into the next. The order is set so that any carryover does the least harm.
- Blood culture bottles. These come first because they must stay sterile.
- Light blue (sodium citrate). Other additives can throw off clotting tests, so this goes before them. A plastic tube with clot activator must not come before it.
- Serum tubes (red, gold). These can be with or without clot activator or gel.
- Green (heparin). This can be with or without gel.
- Lavender or pink (EDTA). EDTA contains potassium and binds calcium, so carryover would falsely raise potassium and lower calcium in any tube filled after it.
- Gray (fluoride and oxalate). These additives also change other test results, so this tube goes last.
A common memory aid is "Boys Love Roses, Growing Lovely Gardens," for blood cultures, light blue, red and gold, green, lavender and gray.
The order is the same for every method. If you draw with a syringe, transfer the blood into the tubes in this same order, using a safety transfer device.
Worked example. An order calls for a CBC, a PT/INR and a basic metabolic panel in a gold tube. Your tubes are lavender, light blue and gold. The order is light blue, gold, lavender.
Filling Tubes
Tubes contain a set amount of additive for a set amount of blood. Let each tube fill until the vacuum stops.
The light blue tube is the strictest. It must be filled to the line, because too little blood leaves too much citrate and gives a false result. If you use a butterfly set and light blue is your first tube, draw a discard tube first. The discard tube clears air from the tubing so the blue tube fills correctly. Your lab's procedure will say whether a discard tube is needed with a straight needle.
Mixing Tubes
Mix each additive tube as soon as you take it off the needle. One inversion means turning the tube upside down and back, gently. Most additive tubes need about 5 to 10 inversions, and light blue tubes often need 3 or 4. Follow the tube maker's directions. Never shake a tube, because shaking breaks red blood cells.
Labeling
Label every tube at the patient's side, right after you collect it, before you leave the patient.
- Confirm the patient's identity with two identifiers.
- Include the patient's full name, date of birth, medical record number if used, the date and time of collection and your initials.
- Check the label against the patient and the order.
Warning: Never label tubes before the draw, and never carry an unlabeled tube away from the patient. A mislabeled specimen can lead to the wrong treatment or a fatal transfusion error.
Collecting Blood Cultures
Skin bacteria in a blood culture can look like a bloodstream infection, which leads to unneeded antibiotics and longer hospital stays.
- Clean the site with chlorhexidine or povidone-iodine, following your procedure, and let it dry fully.
- Clean the bottle tops with alcohol, and let them dry.
- Do not touch the cleaned site again.
- With a butterfly set, fill the aerobic bottle first, so air in the tubing does not enter the anaerobic bottle. Follow your facility's procedure for a syringe draw.
- Fill each bottle with the amount the bottle maker directs, often 8 to 10 mL for adults.
- If two sets are ordered, collect them from separate sites and label each one with its site.
Handling and Transport
Get specimens to the lab quickly. In a tube without a glycolytic inhibitor, glucose falls about 5 to 7 percent each hour at room temperature because blood cells keep using it.
| Handling need | Examples | Why |
|---|---|---|
| Room temperature | Most routine specimens | Standard handling |
| On ice | Ammonia, lactic acid | These break down quickly at room temperature |
| Your lab's procedure | Arterial blood gases | Plastic syringes usually stay at room temperature and reach the analyzer within 30 minutes, because ice can distort the oxygen result |
| Kept warm (37°C) | Cold agglutinins, cryoglobulins, cryofibrinogen | They can drop out of the sample as it cools |
| Protected from light | Bilirubin | Light breaks it down, so wrap the tube in foil |
Keep tubes upright when you can. Handle them gently, and use padded carriers if your site has a pneumatic tube system, because rough travel can cause hemolysis.
Serum tubes need about 30 to 60 minutes to clot before they go in a centrifuge, depending on the tube, so check the maker's instructions. Spinning too early leaves clots in the serum. Tubes with EDTA or heparin do not need clotting time.
Why a Lab Rejects a Specimen
- The label is missing, wrong or does not match the order.
- The specimen is hemolyzed, meaning the serum or plasma looks pink or red.
- It clotted when it should not have.
- It is underfilled, especially a light blue tube.
- It was collected in the wrong tube.
- It was stored at the wrong temperature, or took too long to arrive.
Rejection means a second stick for the patient and a delay in care. If your specimen is rejected, find out why and fix the habit.
Common Mistakes
| Mistake | What it costs |
|---|---|
| Drawing lavender before green or gold | False potassium and calcium results |
| Underfilling a light blue tube | A false clotting result, and likely rejection |
| Shaking tubes to mix them | Hemolysis |
| Labeling tubes at the nurses' station | A risk of mixing up patients |
| Leaving an ammonia tube on the counter | A falsely high result |
Check Your Understanding
1. You need green, gray, light blue and gold tubes. In what order do you fill them?
Light blue, gold, green, gray.
2. You draw with a syringe and need gold and lavender tubes. Which one do you fill first?
Gold. The order is the same for a syringe.
3. Your lactic acid tube is ready, and your cart is full. What do you need to do?
Put it on ice and get it to the lab right away, as your procedure directs.
4. The serum in a tube looks pink after spinning. What happened, and what could have caused it?
It is probably hemolyzed. Possible causes include shaking the tube, pulling too hard on a syringe or forcing blood through a small needle.
Key Takeaways
- The order is blood cultures, light blue, red or gold, green, lavender and gray.
- The order stays the same for syringes and butterfly sets.
- Fill light blue tubes to the line, and invert additive tubes gently.
- Label at the patient's side, right after collection.
- Ammonia and lactic acid go on ice, blood gases follow your lab's procedure, and bilirubin stays out of the light.
- Serum tubes need about 30 to 60 minutes to clot before centrifuging.