3.2 Order of Draw
Key Takeaways
The order of draw prevents cross-contamination of tube additives between tubes, which can cause erroneous test results
The correct order of draw for evacuated tubes is: Blood cultures (yellow/sterile) -> Light blue (citrate) -> Red/Gold (SST/no additive) -> Green (heparin) -> Lavender/Pink (EDTA) -> Gray (sodium fluoride/oxalate)
The mnemonic "Boys Love Roses, Girls Love Grapes" helps remember: Blood cultures, Light blue, Red/Gold, Green, Lavender, Gray
Light blue (citrate) tubes must be filled to the exact fill line — the blood-to-anticoagulant ratio of 9:1 is critical for accurate coagulation results
If a light blue tube is the ONLY tube ordered (no blood culture), it is drawn FIRST — no discard tube is needed with a standard ETS
Tubes with additives must be gently inverted (mixed) 5-10 times immediately after collection — NEVER shake the tubes
SST (serum separator tubes) with gold tops contain a clot activator and gel separator — invert 5 times after collection
EDTA (lavender top) is the additive used for hematology tests (CBC) — it prevents clotting by chelating calcium
Order of Draw — The #1 Tested Topic
The order of draw is the single most important and most frequently tested topic on the NHA CPT exam. It determines the sequence in which evacuated tubes are filled during a venipuncture procedure. Following the correct order prevents cross-contamination of tube additives, which can cause erroneous test results.
Why Order of Draw Matters
When a needle punctures a tube stopper, small amounts of the tube's additive can be carried on the needle into the next tube. For example:
- If an EDTA tube (lavender) is drawn before a citrate tube (light blue), trace amounts of EDTA can contaminate the citrate tube and falsely affect coagulation results
- If a heparin tube (green) is drawn before a serum tube (red), heparin contamination can prevent proper clotting in the serum tube
Following the standardized order of draw eliminates this risk.
The Order of Draw (CLSI Standard)
The Clinical and Laboratory Standards Institute (CLSI) standard PRE02 (8th edition, February 2025; formerly GP41) sets the standard order of draw. The renumbering did not change the order:
| Order | Tube Color | Additive | Common Tests | Inversions |
|---|---|---|---|---|
| 1st | Yellow (SPS) or Sterile bottles | Sodium polyanethol sulfonate (SPS) | Blood cultures | 8-10 times |
| 2nd | Light Blue | Sodium citrate (3.2%) | Coagulation tests (PT, INR, PTT, fibrinogen, D-dimer) | 3-4 times |
| 3rd | Red | Glass: no additive (silicone coated); plastic: clot activator | Serum tests — some chemistry, serology, blood bank | Glass: 0; plastic: 5 times |
| 3rd | Gold (SST) | Clot activator + gel separator | Serum chemistry (BMP, CMP, lipid panel, thyroid, liver panel) | 5 times |
| 4th | Green | Lithium heparin or sodium heparin | Plasma chemistry (stat electrolytes, ammonia, BMP) | 8-10 times |
| 5th | Lavender (Purple) | EDTA (ethylenediaminetetraacetic acid) | Hematology (CBC, differential, ESR, hemoglobin A1c) | 8-10 times |
| 5th | Pink | EDTA | Blood bank (type and screen, crossmatch) | 8-10 times |
| 6th | Gray | Sodium fluoride + potassium oxalate | Glucose, lactate, alcohol levels | 8-10 times |
Memory Aid: "Boys Love Roses, Girls Love Grapes"
| Letter | Color | Tube |
|---|---|---|
| B — Boys | Blood culture bottles | Yellow SPS / sterile bottles |
| L — Love | Light blue | Sodium citrate (coagulation) |
| R — Roses | Red / Gold | No additive / SST (serum) |
| G — Girls | Green | Heparin (plasma) |
| L — Love | Lavender / Pink | EDTA (hematology / blood bank) |
| G — Grapes | Gray | Sodium fluoride (glucose) |
Critical Rules for Order of Draw
Rule 1: Blood Cultures Are ALWAYS First
- Blood cultures must be collected in the most sterile manner possible
- Drawing them first minimizes the risk of contamination from tube additives
- The venipuncture site must be cleaned with chlorhexidine or povidone-iodine (not just alcohol) for blood cultures
- Two sets are typically collected from two separate venipuncture sites
Rule 2: Light Blue Tubes Require Exact Fill Volume
- The blood-to-anticoagulant ratio must be 9:1 (9 parts blood to 1 part citrate)
- Under-filled tubes will have too much citrate relative to blood, causing falsely prolonged coagulation results
- Fill the tube to the exact fill line — if the tube is short, it must be rejected and redrawn
- With a butterfly needle, draw a discard tube first to account for dead space in the tubing
Rule 3: Light Blue as the Only or First Tube
- If light blue is the only tube ordered and you are using a standard ETS (no butterfly), draw it first — no discard tube is needed
- If using a butterfly needle and light blue is the first tube, draw a discard tube (plain or citrate) first to fill the dead space
Rule 4: Invert Tubes Gently — NEVER Shake
- Tubes with additives must be gently inverted (turned upside down and back) the recommended number of times immediately after filling
- Shaking tubes causes hemolysis (destruction of red blood cells), which can invalidate test results
- Plain red-top tubes (no additive) do NOT require inversion
Rule 5: SST Tubes Must Clot Before Centrifugation
- Gold/SST tubes contain a clot activator and require 30 minutes of clotting time at room temperature before centrifugation
- Red-top tubes (no additive) require 60 minutes for complete clotting
Tube Additives and Their Functions
| Additive | Function | Found In |
|---|---|---|
| Sodium citrate (3.2%) | Binds calcium to prevent clotting; preserves coagulation factors | Light blue top |
| Clot activator | Accelerates clotting to produce serum faster | Gold (SST), red/gray (tiger top) |
| Gel separator | Forms a barrier between serum/plasma and cells during centrifugation | Gold (SST), green/gray (PST) |
| Lithium heparin | Inhibits thrombin to prevent clotting; produces plasma | Green top |
| EDTA | Chelates (binds) calcium to prevent clotting; preserves cell morphology | Lavender/purple, pink top |
| Sodium fluoride | Inhibits glycolysis (prevents glucose breakdown); preserves glucose levels | Gray top |
| Potassium oxalate | Binds calcium to prevent clotting | Gray top (combined with sodium fluoride) |
| SPS | Prevents coagulation; inhibits complement and phagocytosis | Yellow top (blood culture) |
Order of Draw — Clinical Scenarios
Understanding the order of draw is critical when you receive multi-tube orders. Here are common clinical scenarios:
Scenario 1: CBC, BMP, and PT/INR
- Tests ordered: CBC (lavender EDTA), BMP (gold SST), PT/INR (light blue citrate)
- Correct order: Light blue -> Gold SST -> Lavender
- Reason: Citrate first (of the ordered tubes), then serum, then EDTA
Scenario 2: Blood Cultures, CBC, and Glucose
- Tests ordered: Blood cultures (yellow/sterile), CBC (lavender EDTA), glucose (gray NaF)
- Correct order: Blood cultures -> Lavender -> Gray
- Reason: Blood cultures always first, EDTA before gray
Scenario 3: PT/INR Only (Using Butterfly Needle)
- Test ordered: PT/INR (light blue citrate) — drawn with butterfly
- Correct order: Discard tube (plain or citrate) -> Light blue
- Reason: Dead space in butterfly tubing must be filled first to ensure proper citrate ratio
Scenario 4: Comprehensive Metabolic Panel and Lipid Panel
- Tests ordered: CMP and lipid panel — both run on serum (gold SST)
- Correct order: One gold SST tube (both tests from same tube)
- Note: Verify the tube volume is sufficient for both panels
Tube Fill Volumes and Inversions — Quick Reference
| Tube Color | Typical Adult Volume | Inversions Required | Special Notes |
|---|---|---|---|
| Yellow (SPS) | 8-10 mL per bottle | 8-10 | Fill to bottle line |
| Light blue | 2.7 mL or 4.5 mL | 3-4 | Must be filled to exact line (9:1 ratio) |
| Red (plain) | 7-10 mL | 0 | No additive; allow 60 min to clot |
| Gold (SST) | 5-8.5 mL | 5 | Clot activator; allow 30 min to clot |
| Green | 4-6 mL | 8-10 | Do not allow to clot |
| Lavender | 3-4 mL | 8-10 | Do not allow to clot |
| Pink | 6 mL | 8-10 | Same as lavender (EDTA) |
| Gray | 4-6 mL | 8-10 | Do not allow to clot |
What is the correct order of draw for the following tubes: Green, Light blue, Lavender, Gold SST?
Gold SST, Green, Light blue, Lavender
Light blue, Gold SST, Green, Lavender
Lavender, Green, Gold SST, Light blue
Green, Light blue, Lavender, Gold SST
Why must a light blue (citrate) tube be filled to the exact fill line?
Under-filling causes hemolysis of red blood cells
The 9:1 blood-to-anticoagulant ratio is critical for accurate coagulation results
Over-filling activates the clotting cascade prematurely
The tube will not fit in the centrifuge if under-filled
Arrange the following tube colors in the correct order of draw:
Arrange the items in the correct order
Light blue (citrate)
Gray (sodium fluoride)
Gold/Red (SST/no additive)
Green (heparin)
Yellow/Sterile (blood cultures)
Lavender (EDTA)
Match each tube color to its additive:
Match each item on the left with the correct item on the right
Light blue
Lavender/Purple
Green
Gray
Gold (SST)
A phlebotomist accidentally draws a lavender (EDTA) tube before a light blue (citrate) tube. What is the potential consequence?
The citrate tube may be contaminated with EDTA, causing inaccurate coagulation results
The EDTA tube will not have enough blood
The citrate tube will clot prematurely
There is no consequence — order of draw is only a suggestion
Which tube additive works by chelating (binding) calcium to prevent clotting and is used for hematology tests like the CBC?
Sodium citrate
Lithium heparin
EDTA (ethylenediaminetetraacetic acid)
Sodium fluoride
The required blood-to-anticoagulant ratio for light blue (citrate) tubes is ___.
Type your answer below
Which tube color contains sodium fluoride and is used for glucose testing?
Green
Lavender
Light blue
Gray
How many times should a lavender (EDTA) tube be inverted after collection?
0 times (do not invert)
3-4 times
5 times
8-10 times
A phlebotomist needs to draw blood for a CBC, PT/INR, and lipid panel. What is the correct order of tubes?
Lavender (CBC), Light blue (PT/INR), Gold SST (lipid panel)
Light blue (PT/INR), Gold SST (lipid panel), Lavender (CBC)
Gold SST (lipid panel), Lavender (CBC), Light blue (PT/INR)
Light blue (PT/INR), Lavender (CBC), Gold SST (lipid panel)
Which of the following tubes contain anticoagulants that work by binding (chelating) calcium? (Select all that apply)
Select all that apply
Light blue (sodium citrate)
Gold SST (clot activator + gel)
Lavender (EDTA)
Green (lithium heparin)
Gray (sodium fluoride + potassium oxalate)
Red (no additive)
What is the mnemonic "Boys Love Roses, Girls Love Grapes" used for in phlebotomy?
Remembering the steps of venipuncture
Remembering the order of draw for evacuated tubes
Remembering the steps of PPE donning
Remembering the types of white blood cells
A phlebotomist collects a light blue (citrate) tube and notices it is only half full. What should they do?
Submit the tube as-is — partial fills are acceptable for citrate tubes
Add saline to bring the tube to the fill line
Reject the tube and recollect a properly filled tube
Shake the tube vigorously to mix the smaller volume with citrate
Which tube color contains a clot activator that accelerates the clotting process?
Lavender (EDTA)
Green (heparin)
Gold (SST)
Light blue (citrate)
Blood cultures are drawn FIRST in the order of draw because:
They require the largest volume of blood
They must be collected under the most sterile conditions to prevent contamination
The SPS additive interferes with other tube additives
They require the warmest specimen temperature
The additive sodium fluoride (found in gray-top tubes) preserves glucose levels by:
Preventing hemolysis of red blood cells
Inhibiting glycolysis (preventing cells from consuming glucose)
Chelating calcium to prevent clotting
Destroying white blood cells that consume glucose
Sections you finish are checked off in the contents.